LOCUS       sequence-354676-        8021 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dual T7 promoter vector for bacterial expression of SpCas9 with a 
            c-terminal NLS and 3xFLAG tag, along with an SpCas9 sgRNA targeting 
            EGFP site 1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8021)
  AUTHORS   Christie KA, Guo JA, Silverstein RA, Doll RM, Mabuchi M, Stutzman 
            HE, Lin J, Ma L, Walton RT, Pinello L, Robb GB, Kleinstiver BP
  TITLE     Precise DNA cleavage using CRISPR-SpRYgests.
  JOURNAL   Nat Biotechnol. 2022 Oct 6. pii: 10.1038/s41587-022-01492-y. doi: 
            10.1038/s41587-022-01492-y.
  PUBMED    36203014
REFERENCE   2  (bases 1 to 8021)
  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: "Nat 
            Biotechnol. 2022 Oct 6. pii: 10.1038/s41587-022-01492-y. doi: 
            10.1038/s41587-022-01492-y."
COMMENT     Sequence Label: pACYC-T7-SpCas9-T7-EGFP_gRNA1-T7term (BPK848)
FEATURES             Location/Qualifiers
     source          1..8021
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     512..531
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(551..628)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        674..692
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    693..717
                     /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)."
     CDS             join(761..4162,4163..4165,4166..4762,4763,4764..4766,
                     4767..4768,4769..4771,4772..4864)
                     /codon_start=1
                     /product="Streptococcus pyogenes Cas9 endonuclease with the
                     D1135V/R1335Q/T1337R mutations"
                     /label=Cas9 VQR
                     /note="has an altered recognition specificity for the 
                     protospacer adjacent motif (PAM) (Kleinstiver et al., 
                     2015)"
                     /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             761..4864
                     /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             761..4864
                     /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             4871..4891
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4898..4963
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     promoter        5033..5051
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        5072..5147
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(5149..5167)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5163..5210
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(5431..6090)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6005..6024
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6091..6193)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(6585..6602)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6719..7264)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(6799..6818)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7489..8021,1..550))
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
       61 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
      121 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
      181 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
      241 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
      301 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
      361 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
      421 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
      481 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
      541 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
      601 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
      661 tgcattagga aattaatacg actcactata ggggaattgt gagcggataa caattcccct
      721 gtagaaataa ttttgtttaa ctttaataag gagatatacc atggataaaa agtattctat
      781 tggtttagac atcggcacta attccgttgg atgggctgtc ataaccgatg aatacaaagt
      841 accttcaaag aaatttaagg tgttggggaa cacagaccgt cattcgatta aaaagaatct
      901 tatcggtgcc ctcctattcg atagtggcga aacggcagag gcgactcgcc tgaaacgaac
      961 cgctcggaga aggtatacac gtcgcaagaa ccgaatatgt tacttacaag aaatttttag
     1021 caatgagatg gccaaagttg acgattcttt ctttcaccgt ttggaagagt ccttccttgt
     1081 cgaagaggac aagaaacatg aacggcaccc catctttgga aacatagtag atgaggtggc
     1141 atatcatgaa aagtacccaa cgatttatca cctcagaaaa aagctagttg actcaactga
     1201 taaagcggac ctgaggttaa tctacttggc tcttgcccat atgataaagt tccgtgggca
     1261 ctttctcatt gagggtgatc taaatccgga caactcggat gtcgacaaac tgttcatcca
     1321 gttagtacaa acctataatc agttgtttga agagaaccct ataaatgcaa gtggcgtgga
     1381 tgcgaaggct attcttagcg cccgcctctc taaatcccga cggctagaaa acctgatcgc
     1441 acaattaccc ggagagaaga aaaatgggtt gttcggtaac cttatagcgc tctcactagg
     1501 cctgacacca aattttaagt cgaacttcga cttagctgaa gatgccaaat tgcagcttag
     1561 taaggacacg tacgatgacg atctcgacaa tctactggca caaattggag atcagtatgc
     1621 ggacttattt ttggctgcca aaaaccttag cgatgcaatc ctcctatctg acatactgag
     1681 agttaatact gagattacca aggcgccgtt atccgcttca atgatcaaaa ggtacgatga
     1741 acatcaccaa gacttgacac ttctcaaggc cctagtccgt cagcaactgc ctgagaaata
     1801 taaggaaata ttctttgatc agtcgaaaaa cgggtacgca ggttatattg acggcggagc
     1861 gagtcaagag gaattctaca agtttatcaa acccatatta gagaagatgg atgggacgga
     1921 agagttgctt gtaaaactca atcgcgaaga tctactgcga aagcagcgga ctttcgacaa
     1981 cggtagcatt ccacatcaaa tccacttagg cgaattgcat gctatactta gaaggcagga
     2041 ggatttttat ccgttcctca aagacaatcg tgaaaagatt gagaaaatcc taacctttcg
     2101 cataccttac tatgtgggac ccctggcccg agggaactct cggttcgcat ggatgacaag
     2161 aaagtccgaa gaaacgatta ctccctggaa ttttgaggaa gttgtcgata aaggtgcgtc
     2221 agctcaatcg ttcatcgaga ggatgaccaa ctttgacaag aatttaccga acgaaaaagt
     2281 attgcctaag cacagtttac tttacgagta tttcacagtg tacaatgaac tcacgaaagt
     2341 taagtatgtc actgagggca tgcgtaaacc cgcctttcta agcggagaac agaagaaagc
     2401 aatagtagat ctgttattca agaccaaccg caaagtgaca gttaagcaat tgaaagagga
     2461 ctactttaag aaaattgaat gcttcgattc tgtcgagatc tccggggtag aagatcgatt
     2521 taatgcgtca cttggtacgt atcatgacct cctaaagata attaaagata aggacttcct
     2581 ggataacgaa gagaatgaag atatcttaga agatatagtg ttgactctta ccctctttga
     2641 agatcgggaa atgattgagg aaagactaaa aacatacgct cacctgttcg acgataaggt
     2701 tatgaaacag ttaaagaggc gtcgctatac gggctgggga cgattgtcgc ggaaacttat
     2761 caacgggata agagacaagc aaagtggtaa aactattctc gattttctaa agagcgacgg
     2821 cttcgccaat aggaacttta tgcagctgat ccatgatgac tctttaacct tcaaagagga
     2881 tatacaaaag gcacaggttt ccggacaagg ggactcattg cacgaacata ttgcgaatct
     2941 tgctggttcg ccagccatca aaaagggcat actccagaca gtcaaagtag tggatgagct
     3001 agttaaggtc atgggacgtc acaaaccgga aaacattgta atcgagatgg cacgcgaaaa
     3061 tcaaacgact cagaaggggc aaaaaaacag tcgagagcgg atgaagagaa tagaagaggg
     3121 tattaaagaa ctgggcagcc agatcttaaa ggagcatcct gtggaaaata cccaattgca
     3181 gaacgagaaa ctttacctct attacctaca aaatggaagg gacatgtatg ttgatcagga
     3241 actggacata aaccgtttat ctgattacga cgtcgatcac attgtacccc aatccttttt
     3301 gaaggacgat tcaatcgaca ataaagtgct tacacgctcg gataagaacc gagggaaaag
     3361 tgacaatgtt ccaagcgagg aagtcgtaaa gaaaatgaag aactattggc ggcagctcct
     3421 aaatgcgaaa ctgataacgc aaagaaagtt cgataactta actaaagctg agaggggtgg
     3481 cttgtctgaa cttgacaagg ccggatttat taaacgtcag ctcgtggaaa cccgccaaat
     3541 cacaaagcat gttgcacaga tactagattc ccgaatgaat acgaaatacg acgagaacga
     3601 taagctgatt cgggaagtca aagtaatcac tttaaagtca aaattggtgt cggacttcag
     3661 aaaggatttt caattctata aagttaggga gataaataac taccaccatg cgcacgacgc
     3721 ttatcttaat gccgtcgtag ggaccgcact cattaagaaa tacccgaagc tagaaagtga
     3781 gtttgtgtat ggtgattaca aagtttatga cgtccgtaag atgatcgcga aaagcgaaca
     3841 ggagataggc aaggctacag ccaaatactt cttttattct aacattatga atttctttaa
     3901 gacggaaatc actctggcaa acggagagat acgcaaacga cctttaattg aaaccaatgg
     3961 ggagacaggt gaaatcgtat gggataaggg ccgggacttc gcgacggtga gaaaagtttt
     4021 gtccatgccc caagtcaaca tagtaaagaa aactgaggtg cagaccggag ggttttcaaa
     4081 ggaatcgatt cttccaaaaa ggaatagtga taagctcatc gctcgtaaaa aggactggga
     4141 cccgaaaaag tacggtggct tcgatagccc tacagttgcc tattctgtcc tagtagtggc
     4201 aaaagttgag aagggaaaat ccaagaaact gaagtcagtc aaagaattat tggggataac
     4261 gattatggag cgctcgtctt ttgaaaagaa ccccatcgac ttccttgagg cgaaaggtta
     4321 caaggaagta aaaaaggatc tcataattaa actaccaaag tatagtctgt ttgagttaga
     4381 aaatggccga aaacggatgt tggctagcgc cggagagctt caaaagggga acgaactcgc
     4441 actaccgtct aaatacgtga atttcctgta tttagcgtcc cattacgaga agttgaaagg
     4501 ttcacctgaa gataacgaac agaagcaact ttttgttgag cagcacaaac attatctcga
     4561 cgaaatcata gagcaaattt cggaattcag taagagagtc atcctagctg atgccaatct
     4621 ggacaaagta ttaagcgcat acaacaagca cagggataaa cccatacgtg agcaggcgga
     4681 aaatattatc catttgttta ctcttaccaa cctcggcgct ccagccgcat tcaagtattt
     4741 tgacacaacg atagatcgca aacgatacac ttctaccaag gaggtgctag acgcgacact
     4801 gattcaccaa tccatcacgg gattatatga aactcggata gatttgtcac agcttggggg
     4861 tgacggatcc cccaagaaga agaggaaagt ctcgagcgac tacaaagacc atgacggtga
     4921 ttataaagat catgacatcg attacaagga tgacgatgac aagtgaagcg gccgcataat
     4981 gcttaagtcg aacagaaagt aatcgtattg tacacggccg cataatcgaa attaatacga
     5041 ctcactatag ggggcacggg cagcttgccg ggttttagag ctagaaatag caagttaaaa
     5101 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgctcc gctgagcaat
     5161 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaacctc
     5221 aggcatttga gaagcacacg gtcacactgc ttccggtagt caataaaccg gtaaaccagc
     5281 aatagacata agcggctatt taacgaccct gccctgaacc gacgaccggg tcgaatttgc
     5341 tttcgaattt ctgccattca tccgcttatt atcacttatt caggcgtagc aaccaggcgt
     5401 ttaagggcac caataactgc cttaaaaaaa ttacgccccg ccctgccact catcgcagta
     5461 ctgttgtaat tcattaagca ttctgccgac atggaagcca tcacaaacgg catgatgaac
     5521 ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta taatatttgc ccatagtgaa
     5581 aacgggggcg aagaagttgt ccatattggc cacgtttaaa tcaaaactgg tgaaactcac
     5641 ccagggattg gctgagacga aaaacatatt ctcaataaac cctttaggga aataggccag
     5701 gttttcaccg taacacgcca catcttgcga atatatgtgt agaaactgcc ggaaatcgtc
     5761 gtggtattca ctccagagcg atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca
     5821 agggtgaaca ctatcccata tcaccagctc accgtctttc attgccatac ggaactccgg
     5881 atgagcattc atcaggcggg caagaatgtg aataaaggcc ggataaaact tgtgcttatt
     5941 tttctttacg gtctttaaaa aggccgtaat atccagctga acggtctggt tataggtaca
     6001 ttgagcaact gactgaaatg cctcaaaatg ttctttacga tgccattggg atatatcaac
     6061 ggtggtatat ccagtgattt ttttctccat tttagcttcc ttagctcctg aaaatctcga
     6121 taactcaaaa aatacgcccg gtagtgatct tatttcatta tggtgaaagt tggaacctct
     6181 tacgtgccga tcaacgtctc attttcgcca aaagttggcc cagggcttcc cggtatcaac
     6241 agggacacca ggatttattt attctgcgaa gtgatcttcc gtcacaggta tttattcggc
     6301 gcaaagtgcg tcgggtgatg ctgccaactt actgatttag tgtatgatgg tgtttttgag
     6361 gtgctccagt ggcttctgtt tctatcagct gtccctcctg ttcagctact gacggggtgg
     6421 tgcgtaacgg caaaagcacc gccggacatc agcgctagcg gagtgtatac tggcttacta
     6481 tgttggcact gatgagggtg tcagtgaagt gcttcatgtg gcaggagaaa aaaggctgca
     6541 ccggtgcgtc agcagaatat gtgatacagg atatattccg cttcctcgct cactgactcg
     6601 ctacgctcgg tcgttcgact gcggcgagcg gaaatggctt acgaacgggg cggagatttc
     6661 ctggaagatg ccaggaagat acttaacagg gaagtgagag ggccgcggca aagccgtttt
     6721 tccataggct ccgcccccct gacaagcatc acgaaatctg acgctcaaat cagtggtggc
     6781 gaaacccgac aggactataa agataccagg cgtttccccc tggcggctcc ctcgtgcgct
     6841 ctcctgttcc tgcctttcgg tttaccggtg tcattccgct gttatggccg cgtttgtctc
     6901 attccacgcc tgacactcag ttccgggtag gcagttcgct ccaagctgga ctgtatgcac
     6961 gaaccccccg ttcagtccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     7021 ccggaaagac atgcaaaagc accactggca gcagccactg gtaattgatt tagaggagtt
     7081 agtcttgaag tcatgcgccg gttaaggcta aactgaaagg acaagttttg gtgactgcgc
     7141 tcctccaagc cagttacctc ggttcaaaga gttggtagct cagagaacct tcgaaaaacc
     7201 gccctgcaag gcggtttttt cgttttcaga gcaagagatt acgcgcagac caaaacgatc
     7261 tcaagaagat catcttatta atcagataaa atatttctag atttcagtgc aatttatctc
     7321 ttcaaatgta gcacctgaag tcagccccat acgatataag ttgtaattct catgttagtc
     7381 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     7441 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     7501 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     7561 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     7621 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     7681 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     7741 ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt
     7801 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     7861 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     7921 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     7981 acgcagacgc gccgagacag aacttaatgg gcccgctaac a
//
