LOCUS       sequence-309987-       13123 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral Cas9 expression, ROSA sgRNA expression, mCherry marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13123)
  TITLE     Lenitviral sgRNA and Cas9 constructs
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 13123)
  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: 
            "Unpublished"
COMMENT     Sequence Label: Cas9 ROSA sgRNA mCherry
FEATURES             Location/Qualifiers
     source          1..13123
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        382..580
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             598..778
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    825..950
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1443..1676
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1861..1905
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2203..2320
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2371..2619
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2371..2391
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2542..2561
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2640..2715
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2777..2988
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3007..3016
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3013..7116
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             3013..7116
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7117..7164
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7165..7188
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7198..7254
                     /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             7255..7962
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(7397..7415)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     7665..7684
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    7978..8566
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8031..8051)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8449..8460)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8638..8871
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(8897..8914)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8903..9127
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9173..9601
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9260..9279)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9470..9491
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9610..9630)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9615..9944
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9795..9930
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9857..9876
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9992..10039
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10058..10432
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    10562..10683
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10599..10618)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10653..10672
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10732..10748)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10732..10748)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10745..10767)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10756..10772
                     /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(10780..10786,10787..10804,10805..10810))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10825..10846
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10963..10980)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11134..11722)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11214..11233)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11893..12684,12685..12753))
                     /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     12516..12535
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12754..12858)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12933..12952)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg
      601 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
      661 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
      721 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
      781 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
      841 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
      901 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
      961 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
     1021 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
     1081 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
     1141 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
     1201 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
     1261 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat
     1321 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
     1381 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
     1441 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
     1501 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
     1561 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
     1621 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
     1681 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
     1741 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
     1801 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
     1861 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
     1921 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
     1981 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
     2041 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa
     2101 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg
     2161 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg
     2221 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
     2281 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
     2341 cagagatcca gtttggttaa ttaaggtacc gagggcctat ttcccatgat tccttcatat
     2401 ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca
     2461 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt
     2521 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat
     2581 ttcttggctt tatatatctt gtggaaagga cgaaacaccg aagatgggcg ggagtcttcg
     2641 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
     2701 gcaccgagtc ggtgcttttt tgaattcgct agctaggtct tgaaaggagt gggaattggc
     2761 tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga
     2821 ggggtcggca attgatccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat
     2881 gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta
     2941 gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacagga ccggttctag
     3001 agcgctgcca ccatggacaa gaagtacagc atcggcctgg acatcggcac caactctgtg
     3061 ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa ggtgctgggc
     3121 aacaccgacc ggcacagcat caagaagaac ctgatcggag ccctgctgtt cgacagcggc
     3181 gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac cagacggaag
     3241 aaccggatct gctatctgca agagatcttc agcaacgaga tggccaaggt ggacgacagc
     3301 ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca cgagcggcac
     3361 cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc caccatctac
     3421 cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct gatctatctg
     3481 gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga cctgaacccc
     3541 gacaacagcg acgtggacaa gctgttcatc cagctggtgc agacctacaa ccagctgttc
     3601 gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc tgccagactg
     3661 agcaagagca gacggctgga aaatctgatc gcccagctgc ccggcgagaa gaagaatggc
     3721 ctgttcggaa acctgattgc cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc
     3781 gacctggccg aggatgccaa actgcagctg agcaaggaca cctacgacga cgacctggac
     3841 aacctgctgg cccagatcgg cgaccagtac gccgacctgt ttctggccgc caagaacctg
     3901 tccgacgcca tcctgctgag cgacatcctg agagtgaaca ccgagatcac caaggccccc
     3961 ctgagcgcct ctatgatcaa gagatacgac gagcaccacc aggacctgac cctgctgaaa
     4021 gctctcgtgc ggcagcagct gcctgagaag tacaaagaga ttttcttcga ccagagcaag
     4081 aacggctacg ccggctacat tgacggcgga gccagccagg aagagttcta caagttcatc
     4141 aagcccatcc tggaaaagat ggacggcacc gaggaactgc tcgtgaagct gaacagagag
     4201 gacctgctgc ggaagcagcg gaccttcgac aacggcagca tcccccacca gatccacctg
     4261 ggagagctgc acgccattct gcggcggcag gaagattttt acccattcct gaaggacaac
     4321 cgggaaaaga tcgagaagat cctgaccttc cgcatcccct actacgtggg ccctctggcc
     4381 aggggaaaca gcagattcgc ctggatgacc agaaagagcg aggaaaccat caccccctgg
     4441 aacttcgagg aagtggtgga caagggcgct tccgcccaga gcttcatcga gcggatgacc
     4501 aacttcgata agaacctgcc caacgagaag gtgctgccca agcacagcct gctgtacgag
     4561 tacttcaccg tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag
     4621 cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt caagaccaac
     4681 cggaaagtga ccgtgaagca gctgaaagag gactacttca agaaaatcga gtgcttcgac
     4741 tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac ataccacgat
     4801 ctgctgaaaa ttatcaagga caaggacttc ctggacaatg aggaaaacga ggacattctg
     4861 gaagatatcg tgctgaccct gacactgttt gaggacagag agatgatcga ggaacggctg
     4921 aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg gcggagatac
     4981 accggctggg gcaggctgag ccggaagctg atcaacggca tccgggacaa gcagtccggc
     5041 aagacaatcc tggatttcct gaagtccgac ggcttcgcca acagaaactt catgcagctg
     5101 atccacgacg acagcctgac ctttaaagag gacatccaga aagcccaggt gtccggccag
     5161 ggcgatagcc tgcacgagca cattgccaat ctggccggca gccccgccat taagaagggc
     5221 atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc
     5281 gagaacatcg tgatcgaaat ggccagagag aaccagacca cccagaaggg acagaagaac
     5341 agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag agctgggcag ccagatcctg
     5401 aaagaacacc ccgtggaaaa cacccagctg cagaacgaga agctgtacct gtactacctg
     5461 cagaatgggc gggatatgta cgtggaccag gaactggaca tcaaccggct gtccgactac
     5521 gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg actccatcga caacaaggtg
     5581 ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg tgccctccga agaggtcgtg
     5641 aagaagatga agaactactg gcggcagctg ctgaacgcca agctgattac ccagagaaag
     5701 ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg aactggataa ggccggcttc
     5761 atcaagagac agctggtgga aacccggcag atcacaaagc acgtggcaca gatcctggac
     5821 tcccggatga acactaagta cgacgagaat gacaagctga tccgggaagt gaaagtgatc
     5881 accctgaagt ccaagctggt gtccgatttc cggaaggatt tccagtttta caaagtgcgc
     5941 gagatcaaca actaccacca cgcccacgac gcctacctga acgccgtcgt gggaaccgcc
     6001 ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt acggcgacta caaggtgtac
     6061 gacgtgcgga agatgatcgc caagagcgag caggaaatcg gcaaggctac cgccaagtac
     6121 ttcttctaca gcaacatcat gaactttttc aagaccgaga ttaccctggc caacggcgag
     6181 atccggaagc ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt gtgggataag
     6241 ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa
     6301 aagaccgagg tgcagacagg cggcttcagc aaagagtcta tcctgcccaa gaggaacagc
     6361 gataagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg cttcgacagc
     6421 cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa
     6481 ctgaagagtg tgaaagagct gctggggatc accatcatgg aaagaagcag cttcgagaag
     6541 aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga cctgatcatc
     6601 aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat gctggcctct
     6661 gccggcgaac tgcagaaggg aaacgaactg gccctgccct ccaaatatgt gaacttcctg
     6721 tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga gcagaaacag
     6781 ctgtttgtgg aacagcacaa gcactacctg gacgagatca tcgagcagat cagcgagttc
     6841 tccaagagag tgatcctggc cgacgctaat ctggacaaag tgctgtccgc ctacaacaag
     6901 caccgggata agcccatcag agagcaggcc gagaatatca tccacctgtt taccctgacc
     6961 aatctgggag cccctgccgc cttcaagtac tttgacacca ccatcgaccg gaagaggtac
     7021 accagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac cggcctgtac
     7081 gagacacgga tcgacctgtc tcagctggga ggcgacaagc gacctgccgc cacaaagaag
     7141 gctggacagg ctaagaagaa gaaagattac aaagacgatg acgataaggg atccggcgca
     7201 acaaacttct ctctgctgaa acaagccgga gatgtcgaag agaatcctgg accggtgagc
     7261 aagggcgagg aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg
     7321 gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac
     7381 gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg
     7441 gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac
     7501 atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac
     7561 ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc
     7621 atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag
     7681 aagaccatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag
     7741 ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag
     7801 accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag
     7861 ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag
     7921 ggccgccact ccaccggcgg catggacgag ctgtacaagt aaacgcgtta agtcgacaat
     7981 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     8041 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     8101 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     8161 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     8221 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     8281 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     8341 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     8401 tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     8461 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     8521 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcgtcg actttaagac
     8581 caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg
     8641 aagggctaat tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct
     8701 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
     8761 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
     8821 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agggcccgtt
     8881 taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     8941 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     9001 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     9061 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     9121 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
     9181 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     9241 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     9301 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     9361 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     9421 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     9481 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     9541 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     9601 taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     9661 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     9721 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     9781 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     9841 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc
     9901 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt
     9961 gtatatccat tttcggatct gatcagcacg tgttgacaat taatcatcgg catagtatat
    10021 cggcatagta taatacgaca aggtgaggaa ctaaaccatg gccaagttga ccagtgccgt
    10081 tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg accggctcgg
    10141 gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg acgtgaccct
    10201 gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct gggtgtgggt
    10261 gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga acttccggga
    10321 cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg agttcgccct
    10381 gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact gacacgtgct
    10441 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
    10501 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
    10561 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
    10621 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
    10681 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
    10741 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
    10801 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
    10861 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
    10921 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
    10981 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
    11041 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
    11101 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
    11161 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
    11221 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
    11281 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
    11341 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
    11401 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
    11461 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
    11521 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
    11581 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
    11641 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
    11701 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
    11761 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
    11821 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
    11881 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
    11941 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
    12001 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
    12061 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
    12121 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
    12181 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
    12241 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
    12301 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
    12361 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
    12421 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
    12481 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
    12541 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
    12601 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
    12661 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
    12721 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
    12781 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
    12841 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg
    12901 agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
    12961 agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt
    13021 taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg
    13081 cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gtt
//
