LOCUS       sequence-142581-       14291 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ubiquitin promoter expresses GFP and humanized spCas9 (from PX330) 
            via a T2A motif. For cell transfection or use in lentiviral 
            packaging. Use Pac1 and/or BstB1 sites to insert sgRNAs from PXL..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14291)
  AUTHORS   Williams MR, Fricano-Kugler CJ, Getz SA, Skelton PD, Lee J, Rizzuto 
            CP, Geller JS, Li M, Luikart BW
  TITLE     A Retroviral CRISPR-Cas9 System for Cellular Autism-Associated 
            Phenotype Discovery in Developing Neurons.
  JOURNAL   Sci Rep. 2016 May 10;6:25611. doi: 10.1038/srep25611.
  PUBMED    27161796
REFERENCE   2  (bases 1 to 14291)
  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: "Sci Rep."; 
            date: "2016-05-10"; volume: "6:25611. doi"; pages: " 
            10.1038/srep25611"
COMMENT     Sequence Label: FUG-T2A-Cas9
FEATURES             Location/Qualifiers
     source          1..14291
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1680..1913
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2098..2142
                     /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    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2649..3859
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3696..3715
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     regulatory      3907..3916
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3913..3915,3916..3918,3919..4629)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3913..3915,3916..3918,3919..4629)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4219..4238)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4566..4587
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4630..4683
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             4690..4755
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4762..4782
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4807..8907
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4807..8907
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             8908..8955
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     misc_feature    8983..9571
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9036..9056)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9454..9465)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(9574..9590)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9575..9591)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             10096..10276
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(10302..10319)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10308..10532
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      10578..11006
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10665..10684)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10875..10896
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(11015..11035)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11020..11349
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11200..11335
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     11262..11281
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        11397..11444
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             11463..11837
                     /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    11967..12088
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12004..12023)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12058..12077
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12137..12153)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12137..12153)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12150..12172)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12161..12177
                     /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(12185..12191,12192..12209,12210..12215))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12230..12251
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12368..12385)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12539..13127)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12619..12638)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13298..14089,14090..14158))
                     /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     13921..13940
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14159..14263)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2581 agatccagtt tggttaatcg catgctagct agttcgaacc ttaattaacc cgtgtcggct
     2641 ccagatctgg cctccgcgcc gggttttggc gcctcccgcg ggcgcccccc tcctcacggc
     2701 gagcgctgcc acgtcagacg aagggcgcag cgagcgtcct gatccttccg cccggacgct
     2761 caggacagcg gcccgctgct cataagactc ggccttagaa ccccagtatc agcagaagga
     2821 cattttagga cgggacttgg gtgactctag ggcactggtt ttctttccag agagcggaac
     2881 aggcgaggaa aagtagtccc ttctcggcga ttctgcggag ggatctccgt ggggcggtga
     2941 acgccgatga ttatataagg acgcgccggg tgtggcacag ctagttccgt cgcagccggg
     3001 atttgggtcg cggttcttgt ttgtggatcg ctgtgatcgt cacttggtga gtagcgggct
     3061 gctgggctgg ccggggcttt cgtggccgcc gggccgctcg gtgggacgga agcgtgtgga
     3121 gagaccgcca agggctgtag tctgggtccg cgagcaaggt tgccctgaac tgggggttgg
     3181 ggggagcgca caaaatggcg gctgttcccg agtcttgaat ggaagacgct tgtaaggcgg
     3241 gctgtgaggt cgttgaaaca aggtgggggg catggtgggc ggcaagaacc caaggtcttg
     3301 aggccttcgc taatgcggga aagctcttat tcgggtgaga tgggctgggg caccatctgg
     3361 ggaccctgac gtgaagtttg tcactgactg gagaactcgg gtttgtcgtc tggttgcggg
     3421 ggcggcagtt atgcggtgcc gttgggcagt gcacccgtac ctttgggagc gcgcgcctcg
     3481 tcgtgtcgtg acgtcacccg ttctgttggc ttataatgca gggtggggcc acctgccggt
     3541 aggtgtgcgg taggcttttc tccgtcgcag gacgcagggt tcgggcctag ggtaggctct
     3601 cctgaatcga caggcgccgg acctctggtg aggggaggga taagtgaggc gtcagtttct
     3661 ttggtcggtt ttatgtacct atcttcttaa gtagctgaag ctccggtttt gaactatgcg
     3721 ctcggggttg gcgagtgtgt tttgtgaagt tttttaggca ccttttgaaa tgtaatcatt
     3781 tgggtcaata tgtaattttc agtgttagac tagtaaattg tccgctaaat tctggccgtt
     3841 tttggctttt ttgttagacg aagcttgggc tgcaggtcga ctctagagga tccccgggta
     3901 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     3961 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     4021 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     4081 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     4141 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     4201 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     4261 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     4321 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     4381 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     4441 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     4501 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     4561 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     4621 ctgtacaagg agggcagagg aagtcttcta acatgcggtg acgtggagga gaatcccggc
     4681 cctgaattcg actataagga ccacgacgga gactacaagg atcatgatat tgattacaaa
     4741 gacgatgacg ataagatggc cccaaagaag aagcggaagg tcggtatcca cggagtccca
     4801 gcagccgaca agaagtacag catcggcctg gacatcggca ccaactctgt gggctgggcc
     4861 gtgatcaccg acgagtacaa ggtgcccagc aagaaattca aggtgctggg caacaccgac
     4921 cggcacagca tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc
     4981 gaggccaccc ggctgaagag aaccgccaga agaagataca ccagacggaa gaaccggatc
     5041 tgctatctgc aagagatctt cagcaacgag atggccaagg tggacgacag cttcttccac
     5101 agactggaag agtccttcct ggtggaagag gataagaagc acgagcggca ccccatcttc
     5161 ggcaacatcg tggacgaggt ggcctaccac gagaagtacc ccaccatcta ccacctgaga
     5221 aagaaactgg tggacagcac cgacaaggcc gacctgcggc tgatctatct ggccctggcc
     5281 cacatgatca agttccgggg ccacttcctg atcgagggcg acctgaaccc cgacaacagc
     5341 gacgtggaca agctgttcat ccagctggtg cagacctaca accagctgtt cgaggaaaac
     5401 cccatcaacg ccagcggcgt ggacgccaag gccatcctgt ctgccagact gagcaagagc
     5461 agacggctgg aaaatctgat cgcccagctg cccggcgaga agaagaatgg cctgttcgga
     5521 aacctgattg ccctgagcct gggcctgacc cccaacttca agagcaactt cgacctggcc
     5581 gaggatgcca aactgcagct gagcaaggac acctacgacg acgacctgga caacctgctg
     5641 gcccagatcg gcgaccagta cgccgacctg tttctggccg ccaagaacct gtccgacgcc
     5701 atcctgctga gcgacatcct gagagtgaac accgagatca ccaaggcccc cctgagcgcc
     5761 tctatgatca agagatacga cgagcaccac caggacctga ccctgctgaa agctctcgtg
     5821 cggcagcagc tgcctgagaa gtacaaagag attttcttcg accagagcaa gaacggctac
     5881 gccggctaca ttgacggcgg agccagccag gaagagttct acaagttcat caagcccatc
     5941 ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg
     6001 cggaagcagc ggaccttcga caacggcagc atcccccacc agatccacct gggagagctg
     6061 cacgccattc tgcggcggca ggaagatttt tacccattcc tgaaggacaa ccgggaaaag
     6121 atcgagaaga tcctgacctt ccgcatcccc tactacgtgg gccctctggc caggggaaac
     6181 agcagattcg cctggatgac cagaaagagc gaggaaacca tcaccccctg gaacttcgag
     6241 gaagtggtgg acaagggcgc ttccgcccag agcttcatcg agcggatgac caacttcgat
     6301 aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga gtacttcacc
     6361 gtgtataacg agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc
     6421 ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg
     6481 accgtgaagc agctgaaaga ggactacttc aagaaaatcg agtgcttcga ctccgtggaa
     6541 atctccggcg tggaagatcg gttcaacgcc tccctgggca cataccacga tctgctgaaa
     6601 attatcaagg acaaggactt cctggacaat gaggaaaacg aggacattct ggaagatatc
     6661 gtgctgaccc tgacactgtt tgaggacaga gagatgatcg aggaacggct gaaaacctat
     6721 gcccacctgt tcgacgacaa agtgatgaag cagctgaagc ggcggagata caccggctgg
     6781 ggcaggctga gccggaagct gatcaacggc atccgggaca agcagtccgg caagacaatc
     6841 ctggatttcc tgaagtccga cggcttcgcc aacagaaact tcatgcagct gatccacgac
     6901 gacagcctga cctttaaaga ggacatccag aaagcccagg tgtccggcca gggcgatagc
     6961 ctgcacgagc acattgccaa tctggccggc agccccgcca ttaagaaggg catcctgcag
     7021 acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc
     7081 gtgatcgaaa tggccagaga gaaccagacc acccagaagg gacagaagaa cagccgcgag
     7141 agaatgaagc ggatcgaaga gggcatcaaa gagctgggca gccagatcct gaaagaacac
     7201 cccgtggaaa acacccagct gcagaacgag aagctgtacc tgtactacct gcagaatggg
     7261 cgggatatgt acgtggacca ggaactggac atcaaccggc tgtccgacta cgatgtggac
     7321 catatcgtgc ctcagagctt tctgaaggac gactccatcg acaacaaggt gctgaccaga
     7381 agcgacaaga accggggcaa gagcgacaac gtgccctccg aagaggtcgt gaagaagatg
     7441 aagaactact ggcggcagct gctgaacgcc aagctgatta cccagagaaa gttcgacaat
     7501 ctgaccaagg ccgagagagg cggcctgagc gaactggata aggccggctt catcaagaga
     7561 cagctggtgg aaacccggca gatcacaaag cacgtggcac agatcctgga ctcccggatg
     7621 aacactaagt acgacgagaa tgacaagctg atccgggaag tgaaagtgat caccctgaag
     7681 tccaagctgg tgtccgattt ccggaaggat ttccagtttt acaaagtgcg cgagatcaac
     7741 aactaccacc acgcccacga cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa
     7801 aagtacccta agctggaaag cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg
     7861 aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta cttcttctac
     7921 agcaacatca tgaacttttt caagaccgag attaccctgg ccaacggcga gatccggaag
     7981 cggcctctga tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa gggccgggat
     8041 tttgccaccg tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa aaagaccgag
     8101 gtgcagacag gcggcttcag caaagagtct atcctgccca agaggaacag cgataagctg
     8161 atcgccagaa agaaggactg ggaccctaag aagtacggcg gcttcgacag ccccaccgtg
     8221 gcctattctg tgctggtggt ggccaaagtg gaaaagggca agtccaagaa actgaagagt
     8281 gtgaaagagc tgctggggat caccatcatg gaaagaagca gcttcgagaa gaatcccatc
     8341 gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat caagctgcct
     8401 aagtactccc tgttcgagct ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa
     8461 ctgcagaagg gaaacgaact ggccctgccc tccaaatatg tgaacttcct gtacctggcc
     8521 agccactatg agaagctgaa gggctccccc gaggataatg agcagaaaca gctgtttgtg
     8581 gaacagcaca agcactacct ggacgagatc atcgagcaga tcagcgagtt ctccaagaga
     8641 gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa gcaccgggat
     8701 aagcccatca gagagcaggc cgagaatatc atccacctgt ttaccctgac caatctggga
     8761 gcccctgccg ccttcaagta ctttgacacc accatcgacc ggaagaggta caccagcacc
     8821 aaagaggtgc tggacgccac cctgatccac cagagcatca ccggcctgta cgagacacgg
     8881 atcgacctgt ctcagctggg aggcgacaaa aggccggcgg ccacgaaaaa ggccggccag
     8941 gcaaaaaaga aaaagtaaga attcgatatc aagcttatcg ataatcaacc tctggattac
     9001 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     9061 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     9121 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     9181 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     9241 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
     9301 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     9361 gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg
     9421 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     9481 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     9541 agtcggatct ccctttgggc cgcctccccg catcgatacc gtcgacctcg agacctagaa
     9601 aaacatggag caatcacaag tagcaataca gcagctacca atgctgattg tgcctggcta
     9661 gaagcacaag aggaggagga ggtgggtttt ccagtcacac ctcaggtacc tttaagacca
     9721 atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa
     9781 gggctaattc actcccaacg aagacaagat atccttgatc tgtggatcta ccacacacaa
     9841 ggctacttcc ctgattggca gaactacaca ccagggccag ggatcagata tccactgacc
     9901 tttggatggt gctacaagct agtaccagtt gagcaagaga aggtagaaga agccaatgaa
     9961 ggagagaaca cccgcttgtt acaccctgtg agcctgcatg ggatggatga cccggagaga
    10021 gaagtattag agtggaggtt tgacagccgc ctagcatttc atcacatggc ccgagagctg
    10081 catccggact gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc
    10141 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
    10201 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
    10261 tggaaaatct ctagcagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag
    10321 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
    10381 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
    10441 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
    10501 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
    10561 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
    10621 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
    10681 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
    10741 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
    10801 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
    10861 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
    10921 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
    10981 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
    11041 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
    11101 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
    11161 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
    11221 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
    11281 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
    11341 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca gcacgtgttg
    11401 acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg aggaactaaa
    11461 ccatggccaa gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg
    11521 tcgagttctg gaccgaccgg ctcgggttct cccgggactt cgtggaggac gacttcgccg
    11581 gtgtggtccg ggacgacgtg accctgttca tcagcgcggt ccaggaccag gtggtgccgg
    11641 acaacaccct ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg
    11701 aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc catgaccgag atcggcgagc
    11761 agccgtgggg gcgggagttc gccctgcgcg acccggccgg caactgcgtg cacttcgtgg
    11821 ccgaggagca ggactgacac gtgctacgag atttcgattc caccgccgcc ttctatgaaa
    11881 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
    11941 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
    12001 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
    12061 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
    12121 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
    12181 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
    12241 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
    12301 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
    12361 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
    12421 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
    12481 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
    12541 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
    12601 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
    12661 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
    12721 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
    12781 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
    12841 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
    12901 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
    12961 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
    13021 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
    13081 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
    13141 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
    13201 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
    13261 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
    13321 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
    13381 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
    13441 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
    13501 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
    13561 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
    13621 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
    13681 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
    13741 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
    13801 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
    13861 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
    13921 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
    13981 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
    14041 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
    14101 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
    14161 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
    14221 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
    14281 tgccacctga c
//
