LOCUS       sequence-389163-       12815 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dCas9-10XGcn4-P2A-mCherry expression construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12815)
  AUTHORS   Hamilton MC, Fife JD, Akinci E, Yu T, Khowpinitchai B, Cha M, Barkal
            S, Thi TT, Yeo GHT, Barroso JPR, Francoeur MJ, Velimirovic M, 
            Gifford DK, Lettre G, Yu H, Cassa CA, Sherwood RI
  TITLE     Systematic elucidation of genetic mechanisms underlying cholesterol 
            uptake.
  JOURNAL   bioRxiv. 2023 Jan 10:2023.01.09.500804. doi: 
            10.1101/2023.01.09.500804. Preprint.
  PUBMED    36711952
REFERENCE   2  (bases 1 to 12815)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv. 
            2023 Jan 10:2023.01.09.500804. doi: 10.1101/2023.01.09.500804. 
            Preprint."
COMMENT     Sequence Label: p2T CAG dCas9-10XGcn4-P2A-mCherry BlastR
FEATURES             Location/Qualifiers
     source          1..12815
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(34..461)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(144..165)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     356..375
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     588..610
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     602..619
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     603..619
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(678..689,690..852)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     regulatory      877..886
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(892..925)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        940..1319
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1321..1598
                     /label=chicken beta-actin promoter
     intron          1599..2615
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2539..2561
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2623..2642
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2755..2781
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2785..2805
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2815..2838,2839..2841,2842..5397,5398..5400,
                     5401..6915)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the N863A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(2815..2838,2839..2841,2842..5328,5329..5331,
                     5332..6915)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6919..6939
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6973..6999
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             6979..7005
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             7018..7038
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7045..7065
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7129..7185
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             7201..7257
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             7273..7329
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             7345..7401
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             7417..7473
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             7489..7545
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             7561..7617
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             7633..7689
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             7705..7761
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             7777..7833
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             7915..7935
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7942..7998
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             8005..8712
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(8150..8168)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     8418..8437
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(8733..8757)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             8810..8851
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             8861..8878
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(8915..8931)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8915..8931)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     polyA_signal    8955..9076
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    9240..9361
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(9519..9917)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     promoter        complement(9936..9983)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(10031..10360)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10045..10180
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(10099..10118)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     10345..10365
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     misc_feature    join(10375..10562,10563..10574)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(10631..10651)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(10631..10649)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(10670..10686)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10670..10686)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10683..10705)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10694..10710
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(10718..10724,10725..10742,10743..10748))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10763..10784
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10901..10918)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11072..11660)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11152..11171)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11831..12622,12623..12691))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     12454..12473
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12692..12796)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
       61 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
      121 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
      181 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
      241 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
      301 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
      361 agaaagcgaa aggagcgggc gctagggcgc ggcgggtgtg gtggtcacgc tgcgcgtaac
      421 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
      481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      541 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      601 ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac
      661 cggcatatgg ttcttgacag aggtgtaaaa agtactcaaa aattttactc aagtgaaagt
      721 acaagtactt agggaaaatt ttactcaatt aaaagtaaaa gtatctggct agaatcttac
      781 ttgagtaaaa gtaaaaaagt actccattaa aattgtactt gagtattaag gaagtaaaag
      841 taaaagcaag aaagatcgat ctcgaaggat ctggaggcca ccatggtgtc gataacttcg
      901 tatagcatac attatacgaa gttatcgata agcttgctcg acattgatta ttgactagtt
      961 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
     1021 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     1081 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
     1141 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
     1201 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
     1261 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
     1321 tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc ccacccccaa
     1381 ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg gggggggggg
     1441 ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg cggggcgagg cggagaggtg
     1501 cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc
     1561 ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc
     1621 gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt
     1681 tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagcgcttgg
     1741 tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag ccttgagggg ctccgggagg
     1801 gccctttgtg cggggggagc ggctcggggg gtgcgtgcgt gtgtgtgtgc gtggggagcg
     1861 ccgcgtgcgg ctccgcgctg cccggcggct gtgagcgctg cgggcgcggc gcggggcttt
     1921 gtgcgctccg cagtgtgcgc gaggggagcg cggccggggg cggtgccccg cggtgcgggg
     1981 ggggctgcga ggggaacaaa ggctgcgtgc ggggtgtgtg cgtggggggg tgagcagggg
     2041 gtgtgggcgc gtcggtcggg ctgcaacccc ccctgcaccc ccctccccga gttgctgagc
     2101 acggcccggc ttcgggtgcg gggctccgta cggggcgtgg cgcggggctc gccgtgccgg
     2161 gcggggggtg gcggcaggtg ggggtgccgg gcggggcggg gccgcctcgg gccggggagg
     2221 gctcggggga ggggcgcggc ggcccccgga gcgccggcgg ctgtcgaggc gcggcgagcc
     2281 gcagccattg ccttttatgg taatcgtgcg agagggcgca gggacttcct ttgtcccaaa
     2341 tctgtgcgga gccgaaatct gggaggcgcc gccgcacccc ctctagcggg cgcggggcga
     2401 agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc
     2461 gccgtcccct tctccctctc cagcctcggg gctgtccgcg gggggacggc tgccttcggg
     2521 ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac cggcggctct agagcctctg
     2581 ctaaccatgt tcatgccttc ttctttttcc tacagctcct gggcaacgtg ctggttattg
     2641 tgctgtctca tcattttggc aaagaattcc tcgagcggcc gccagtgtga tggatatctg
     2701 cagaattcca gcacactggc ggccgttact agtggatcca ccggtgccac catgtaccca
     2761 tacgatgttc cagattacgc ttcgccgaag aaaaagcgca aggtcgaagc gtccgacaag
     2821 aagtacagca tcggcctggc catcggcacc aactctgtgg gctgggccgt gatcaccgac
     2881 gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc
     2941 aagaagaacc tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg
     3001 ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa
     3061 gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag
     3121 tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg
     3181 gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg
     3241 gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag
     3301 ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag
     3361 ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc
     3421 agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa
     3481 aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggcaa cctgattgcc
     3541 ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa
     3601 ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc
     3661 gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc
     3721 gacatcctga gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag
     3781 agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg
     3841 cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt
     3901 gacggcggag ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg
     3961 gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg
     4021 accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg
     4081 cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc
     4141 ctgaccttcc gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc
     4201 tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac
     4261 aagggcgctt ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc
     4321 aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag
     4381 ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag
     4441 cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag
     4501 ctgaaagagg actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg
     4561 gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac
     4621 aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg
     4681 acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc
     4741 gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc
     4801 cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg
     4861 aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc
     4921 tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac
     4981 attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg
     5041 gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg
     5101 gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg
     5161 atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac
     5221 acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac
     5281 gtggaccagg aactggacat caaccggctg tccgactacg atgtggacgc catcgtgcct
     5341 cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac
     5401 cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg
     5461 cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc
     5521 gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa
     5581 acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac
     5641 gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg
     5701 tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac
     5761 gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag
     5821 ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc
     5881 aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg
     5941 aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc
     6001 gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg
     6061 cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc
     6121 ggcttcagca aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag
     6181 aaggactggg accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg
     6241 ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg
     6301 ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa
     6361 gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg
     6421 ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga
     6481 aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag
     6541 aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag
     6601 cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc
     6661 gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga
     6721 gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc
     6781 ttcaagtact ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg
     6841 gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct
     6901 cagctgggag gcgacagccc caagaagaag agaaaggtgg aggccagcgg gccggccgga
     6961 tccgggcgcg cctatcccta tgacgtgccc gattatgcca gcctgggcag cggctccccc
     7021 aagaaaaaac gcaaggtgga agatcctaag aaaaagcgga aagtggacgg cattggtagt
     7081 gggagcaacg gcagcagcgg atccaacggt ccgactgacg ccgcggaaga agaacttttg
     7141 agcaagaatt atcatcttga gaacgaagtg gctcgtctta agaaaggttc tggcagtgga
     7201 gaagaactgc tttcaaagaa ttaccacctg gaaaatgagg tagctagact gaaaaagggg
     7261 agcggaagtg gggaggagtt gctgagcaaa aattatcatt tggagaacga agtagcacga
     7321 ctaaagaaag ggtccggatc gggtgaggag ttactctcga aaaattatca tctcgaaaac
     7381 gaagtggctc ggctaaaaaa gggcagtggt tctggagaag agctattatc taaaaactac
     7441 cacctcgaaa atgaggtggc acgcttaaaa aagggaagtg gcagtggtga agagctacta
     7501 tccaagaatt atcatcttga gaacgaggta gcgcgtttga agaagggttc cggctcagga
     7561 gaggaactgc tctcgaagaa ctatcatctt gaaaatgagg tcgctcgatt aaaaaaggga
     7621 tcgggcagtg gtgaggaact actttcaaag aattaccacc tcgaaaacga agtagctcga
     7681 ttaaagaaag gttcagggtc gggtgaagaa ttactgagta aaaattatca tctggaaaat
     7741 gaggtagcga gactaaaaaa ggggagtggt tctggcgagg aattgctatc gaaaaattat
     7801 catcttgaga acgaagttgc taggctcaaa aagggctcag gctcaggcac cgcggtaaac
     7861 ataggtggtg gaaccggtcc gatggatcta cagcggccgc aaggtggagg tggacccaag
     7921 aagaagcgca aggtggctag cgctactaac ttcagcctgc tgaagcaggc tggagacgtg
     7981 gaggagaacc ctggacctac gcgtatggtg agcaagggcg aggaggataa catggccatc
     8041 atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc
     8101 gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag
     8161 gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac
     8221 ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc
     8281 cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg
     8341 acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc
     8401 aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc
     8461 gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg
     8521 aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg
     8581 cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac
     8641 tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac
     8701 gagctgtaca agatcgttta attaagaatt cgacccagct ttcttgtaca aagtggttga
     8761 tatccagcac agtggcggcc gctcgagtct agagggcccg cggttcgaag gtaagcctat
     8821 ccctaaccct ctcctcggtc tcgattctac gcgtaccggt catcatcacc atcaccattg
     8881 agtttatcca tcacactggc ggccgctcga gggggatcca ctagttctag agcggccgct
     8941 tcgagcagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg
     9001 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     9061 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga
     9121 gatgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa tcgcgataag
     9181 gatccgggct ggcgtaatag cgaagaggcc cgcaccgggc cgctcgaata cagacatgat
     9241 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
     9301 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     9361 tggggtgggc gaagaactcc agcatgagat ccccgcgctg gaggatcatc cagccggcgt
     9421 cccggaaaac gattccgaag cccaaccttt catagaaggc ggcggtggaa tcgaaatctc
     9481 gtagcacgtg tcagtcctgc tcctcggcca cgaagtgctt agccctccca cacataacca
     9541 gagggcagca attcacgaat cccaactgcc gtcggctgtc catcactgtc cttcactatg
     9601 gctttgatcc caggatgcag atcgagaagc acctgtcggc accgtccgca ggggctcaag
     9661 atgcccctgt tctcatttcc gatcgcgacg atacaagtca ggttgccagc tgccgcagca
     9721 gcagcagtgc ccagcaccac gagttctgca caaggtcccc cagtaaaatg atatacattg
     9781 acaccagtga agatgcggcc gtcgctagag agagctgcgc tggcgacgct gtagtcttca
     9841 gagatgggga tgctgttgat tgtagccgtt gctctttcaa tgagggtgga ttcttcttga
     9901 gacaaaggct tggccatggt ttagttcctc accttgtcgt attatactat gccgatatac
     9961 tatgccgatg attaattgtc aacacgtgct gatcagatcc gaaaatggat atacaagctc
    10021 ccgggagctt tttgcaaaag cctaggcctc caaaaaagcc tcctcactac ttctggaata
    10081 gctcagaggc agaggcggcc tcggcctctg cataaataaa aaaaattagt cagccatggg
    10141 gcggagaatg ggcggaactg ggcggagtta ggggcgggat gggcggagtt aggggcggga
    10201 ctatggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg
    10261 gggactttcc acacctggtt gctgactaat tgagatgcat gctttgcata cttctgcctg
    10321 ctggggagcc tggggacttt ccacacccta actgacacac attccacaga attcaagtga
    10381 tctccaaaaa ataagtactt tttgactgta aataaaattg taaggagtaa aaagtacttt
    10441 tttttctaaa aaaatgtaat taagtaaaag taaaagtatt gatttttaat tgtactcaag
    10501 taaagtaaaa atccccaaaa ataatactta agtacagtaa tcaagtaaaa ttactcaagt
    10561 actttacacc tctggttctt gaccccctac cttcagcaag cccagcagat ccgagctcca
    10621 gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt
    10681 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
    10741 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
    10801 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
    10861 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
    10921 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
    10981 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
    11041 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
    11101 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
    11161 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
    11221 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
    11281 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
    11341 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
    11401 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
    11461 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
    11521 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
    11581 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
    11641 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
    11701 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
    11761 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
    11821 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
    11881 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
    11941 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
    12001 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
    12061 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
    12121 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
    12181 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
    12241 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
    12301 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
    12361 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
    12421 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
    12481 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
    12541 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
    12601 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
    12661 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
    12721 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
    12781 aaataggggt tccgcgcaca tttccccgaa aagtg
//
