LOCUS       sequence-376657-       11667 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express Cas9 in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11667)
  AUTHORS   Lopez SC, Crawford KD, Lear SK, Bhattarai-Kline S, Shipman SL
  TITLE     Precise genome editing across kingdoms of life using retron-derived 
            DNA.
  JOURNAL   Nat Chem Biol. 2022 Feb;18(2):199-206. doi: 
            10.1038/s41589-021-00927-y. Epub 2021 Dec 23.
  PUBMED    34949838
REFERENCE   2  (bases 1 to 11667)
  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"; doi: 
            "10.1038/s41589-021-00927-y"; journalName: "Nat Chem Biol"; date: 
            "2022-02"; volume: "18"; issue: "2"; pages: "199-206"
COMMENT     Sequence Label: pSCL.140
FEATURES             Location/Qualifiers
     source          1..11667
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(131..586)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(268..289)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     480..499
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     713..735
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     727..744
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     728..744
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   874..936
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1385..1763
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    1393..1411
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1429..1447
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1465..1483
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1501..1519
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1537..1555
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1573..1591
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1609..1627
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1672..1696
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1798..1818
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1819..5922
                     /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             1819..5919
                     /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             1819..5919
                     /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"
     primer_bind     complement(5958..5977)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6002..6050
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        6281..6492
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             6505..6773
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             6807..7550
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             7551..7604
                     /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             7605..8204
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(7605..7624)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     8101..8121
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    8237..8358
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8274..8293)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8328..8347
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   9121..9155
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(9646..9662)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9646..9662)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9659..9681)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9670..9686
                     /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(9694..9700,9701..9718,9719..9724))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9739..9760
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9877..9894)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10048..10636)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10128..10147)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10807..11598,11599..11667))
                     /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     11430..11449
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
      181 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
      241 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
      301 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
      361 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
      421 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
      481 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
      541 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc
      601 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
      661 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
      721 cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg
      781 gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg
      841 ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg
      901 tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag
      961 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata
     1021 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaac aaaaactcaa
     1081 aatttcttct ataaagtaac aaaactttta tgagggacag ccccccccca aagcccccag
     1141 ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc
     1201 cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg
     1261 ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga
     1321 cacctggggg gatacgggga aaaggcctcc acggccacta gtctttcgtc ttcaagaatt
     1381 cctcgagttt actccctatc agtgatagag aacgtatgaa gagtttactc cctatcagtg
     1441 atagagaacg tatgcagact ttactcccta tcagtgatag agaacgtata aggagtttac
     1501 tccctatcag tgatagagaa cgtatgacca gtttactccc tatcagtgat agagaacgta
     1561 tctacagttt actccctatc agtgatagag aacgtatatc cagtttactc cctatcagtg
     1621 atagagaacg tataagcttt aggcgtgtac ggtgggcgcc tataaaagca gagctcgttt
     1681 agtgaaccgt cagatcgcct ggagcaattc cacaacactt ttgtcttata ccaactttcc
     1741 gtaccacttc ctaccctcgt aaaggtctag agctaggcca ccatgagcag ggctgacccc
     1801 aagaagaaga ggaaggtgga caagaagtac tccattgggc tcgatatcgg cacaaacagc
     1861 gtcggttggg ccgtcattac ggacgagtac aaggtgccga gcaaaaaatt caaagttctg
     1921 ggcaataccg atcgccacag cataaagaag aacctcattg gcgccctcct gttcgactcc
     1981 ggggagacgg ccgaagccac gcggctcaaa agaacagcac ggcgcagata tacccgcaga
     2041 aagaatcgga tctgctacct gcaggagatc tttagtaatg agatggctaa ggtggatgac
     2101 tctttcttcc ataggctgga ggagtccttt ttggtggagg aggataaaaa gcacgagcgc
     2161 cacccaatct ttggcaatat cgtggacgag gtggcgtacc atgaaaagta cccaaccata
     2221 tatcatctga ggaagaagct tgtagacagt actgataagg ctgacttgcg gttgatctat
     2281 ctcgcgctgg cgcatatgat caaatttcgg ggacacttcc tcatcgaggg ggacctgaac
     2341 ccagacaaca gcgatgtcga caaactcttt atccaactgg ttcagactta caatcagctt
     2401 ttcgaagaga acccgatcaa cgcatccgga gttgacgcca aagcaatcct gagcgctagg
     2461 ctgtccaaat cccggcggct cgaaaacctc atcgcacagc tccctgggga gaagaagaac
     2521 ggcctgtttg gtaatcttat cgccctgtca ctcgggctga cccccaactt taaatctaac
     2581 ttcgacctgg ccgaagatgc caagcttcaa ctgagcaaag acacctacga tgatgatctc
     2641 gacaatctgc tggcccagat cggcgaccag tacgcagacc tttttttggc ggcaaagaac
     2701 ctgtcagacg ccattctgct gagtgatatt ctgcgagtga acacggagat caccaaagct
     2761 ccgctgagcg ctagtatgat caagcgctat gatgagcacc accaagactt gactttgctg
     2821 aaggcccttg tcagacagca actgcctgag aagtacaagg aaattttctt cgatcagtct
     2881 aaaaatggct acgccggata cattgacggc ggagcaagcc aggaggaatt ttacaaattt
     2941 attaagccca tcttggaaaa aatggacggc accgaggagc tgctggtaaa gcttaacaga
     3001 gaagatctgt tgcgcaaaca gcgcactttc gacaatggaa gcatccccca ccagattcac
     3061 ctgggcgaac tgcacgctat cctcaggcgg caagaggatt tctacccctt tttgaaagat
     3121 aacagggaaa agattgagaa aatcctcaca tttcggatac cctactatgt aggccccctc
     3181 gcccggggaa attccagatt cgcgtggatg actcgcaaat cagaagagac catcactccc
     3241 tggaacttcg aggaagtcgt ggataagggg gcctctgccc agtccttcat cgaaaggatg
     3301 actaactttg ataaaaatct gcctaacgaa aaggtgcttc ctaaacactc tctgctgtac
     3361 gagtacttca cagtttataa cgagctcacc aaggtcaaat acgtcacaga agggatgaga
     3421 aagccagcat tcctgtctgg agagcagaag aaagctatcg tggacctcct cttcaagacg
     3481 aaccggaaag ttaccgtgaa acagctcaaa gaagactatt tcaaaaagat tgaatgtttc
     3541 gactctgttg aaatcagcgg agtggaggat cgcttcaacg catccctggg aacgtatcac
     3601 gatctcctga aaatcattaa agacaaggac ttcctggaca atgaggagaa cgaggacatt
     3661 cttgaggaca ttgtcctcac ccttacgttg tttgaagata gggagatgat tgaagaacgc
     3721 ttgaaaactt acgctcatct cttcgacgac aaagtcatga aacagctcaa gaggcgccga
     3781 tatacaggat gggggcggct gtcaagaaaa ctgatcaatg ggatccgaga caagcagagt
     3841 ggaaagacaa tcctggattt tcttaagtcc gatggatttg ccaaccggaa cttcatgcag
     3901 ttgatccatg atgactctct cacctttaag gaggacatcc agaaagcaca agtttctggc
     3961 cagggggaca gtcttcacga gcacatcgct aatcttgcag gtagcccagc tatcaaaaag
     4021 ggaatactgc agaccgttaa ggtcgtggat gaactcgtca aagtaatggg aaggcataag
     4081 cccgagaata tcgttatcga gatggcccga gagaaccaaa ctacccagaa gggacagaag
     4141 aacagtaggg aaaggatgaa gaggattgaa gagggtataa aagaactggg gtcccaaatc
     4201 cttaaggaac acccagttga aaacacccag cttcagaatg agaagctcta cctgtactac
     4261 ctgcagaacg gcagggacat gtacgtggat caggaactgg acatcaatcg gctctccgac
     4321 tacgacgtgg atcatatcgt gccccagtct tttctcaaag atgattctat tgataataaa
     4381 gtgttgacaa gatccgataa aaatagaggg aagagtgata acgtcccctc agaagaagtt
     4441 gtcaagaaaa tgaaaaatta ttggcggcag ctgctgaacg ccaaactgat cacacaacgg
     4501 aagttcgata atctgactaa ggctgaacga ggtggcctgt ctgagttgga taaagccggc
     4561 ttcatcaaaa ggcagcttgt tgagacacgc cagatcacca agcacgtggc ccaaattctc
     4621 gattcacgca tgaacaccaa gtacgatgaa aatgacaaac tgattcgaga ggtgaaagtt
     4681 attactctga agtctaagct ggtctcagat ttcagaaagg actttcagtt ttataaggtg
     4741 agagagatca acaattacca ccatgcgcat gatgcctacc tgaatgcagt ggtaggcact
     4801 gcacttatca aaaaatatcc caagcttgaa tctgaatttg tttacggaga ctataaagtg
     4861 tacgatgtta ggaaaatgat cgcaaagtct gagcaggaaa taggcaaggc caccgctaag
     4921 tacttctttt acagcaatat tatgaatttt ttcaagaccg agattacact ggccaatgga
     4981 gagattcgga agcgaccact tatcgaaaca aacggagaaa caggagaaat cgtgtgggac
     5041 aagggtaggg atttcgcgac agtccggaag gtcctgtcca tgccgcaggt gaacatcgtt
     5101 aaaaagaccg aagtacagac cggaggcttc tccaaggaaa gtatcctccc gaaaaggaac
     5161 agcgacaagc tgatcgcacg caaaaaagat tgggacccca agaaatacgg cggattcgat
     5221 tctcctacag tcgcttacag tgtactggtt gtggccaaag tggagaaagg gaagtctaaa
     5281 aaactcaaaa gcgtcaagga actgctgggc atcacaatca tggagcgatc aagcttcgaa
     5341 aaaaacccca tcgactttct cgaggcgaaa ggatataaag aggtcaaaaa agacctcatc
     5401 attaagcttc ccaagtactc tctctttgag cttgaaaacg gccggaaacg aatgctcgct
     5461 agtgcgggcg agctgcagaa aggtaacgag ctggcactgc cctctaaata cgttaatttc
     5521 ttgtatctgg ccagccacta tgaaaagctc aaagggtctc ccgaagataa tgagcagaag
     5581 cagctgttcg tggaacaaca caaacactac cttgatgaga tcatcgagca aataagcgaa
     5641 ttctccaaaa gagtgatcct cgccgacgct aacctcgata aggtgctttc tgcttacaat
     5701 aagcacaggg ataagcccat cagggagcag gcagaaaaca ttatccactt gtttactctg
     5761 accaacttgg gcgcgcctgc agccttcaag tacttcgaca ccaccataga cagaaagcgg
     5821 tacacctcta caaaggaggt cctggacgcc acactgattc atcagtcaat tacggggctc
     5881 tatgaaacaa gaatcgacct ctctcagctc ggtggagact gaccggttag taatgagttt
     5941 aaacggggga ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg
     6001 acggcaataa aaagacagaa taaaacgcac gggtgttggg tcgtttgttc ataaacgcgg
     6061 ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata
     6121 cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct
     6181 cgcagccaac gtcggggcgg caggccctgc catagcagat ctgcgaaatc ggatccagct
     6241 gaatctaagt ggatctgcga tcgctccggt gcccgtcagt gggcagagcg cacatcgccc
     6301 acagtccccg agaagttggg gggaggggtc ggcaattgaa cgggtgccta gagaaggtgg
     6361 cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg
     6421 ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc
     6481 gccagaacac agctgaagct tcgaggggct cgcatctctc cttcacgcgc ccgccgccct
     6541 acctgaggcc gccatccacg ccggttgagt cgcgttctgc cgcctcccgc ctgtggtgcc
     6601 tcctgaactg cgtccgccgt ctaggtaagt ttaaagctca ggtcgagacc gggcctttgt
     6661 ccggcgctcc cttggagcct acctagactc agccggctct ccacgctttg cctgaccctg
     6721 cttgctcaac tctacgtctt tgtttcgttt tctgttctgc gccgttacag atctaagctg
     6781 tgaccggcgc ctactctaga gccgccatgt ctagactgga caagagcaaa gtcataaact
     6841 ctgctctgga attactcaat ggagtcggta tcgaaggcct gacgacaagg aaactcgctc
     6901 aaaagctggg agttgagcag cctaccctgt actggcacgt gaagaacaag cgggccctgc
     6961 tcgatgccct gccaatcgag atgctggaca ggcatcatac ccactcctgc cccctggaag
     7021 gcgagtcatg gcaagacttt ctgcggaaca acgccaagtc ataccgctgt gctctcctct
     7081 cacatcgcga cggggctaaa gtgcatctcg gcacccgccc aacagagaaa cagtacgaaa
     7141 ccctggaaaa tcagctcgcg ttcctgtgtc agcaaggctt ctccctggag aacgcactgt
     7201 acgctctgtc cgccgtgggc cactttacac tgggctgcgt attggaggaa caggagcatc
     7261 aagtagcaaa agaggaaaga gagacaccta ccaccgattc tatgccccca cttctgaaac
     7321 aagcaattga gctgttcgac cggcagggag ccgaacctgc cttccttttc ggcctggaac
     7381 taatcatatg tggcctggag aaacagctaa agtgcgaaag cggcgggccg accgacgccc
     7441 ttgacgattt tgacttagac atgctcccag ccgatgccct tgacgacttt gaccttgata
     7501 tgctgcctgc tgacgctctt gacgattttg accttgacat gctccccggg gagggcagag
     7561 gaagtcttct aacatgcggt gacgtggagg agaatcccgg ccctatgacc gagtacaagc
     7621 ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg
     7681 cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg
     7741 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg
     7801 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg
     7861 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc
     7921 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg
     7981 ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc
     8041 ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc
     8101 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg
     8161 aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgaaatcaa cctctggatt
     8221 acaaaatttg tgggtaaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     8281 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     8341 actcatcaat gtatcttatc atgtctggaa ttgactcaaa tgatgtcaat tagtctatca
     8401 gaagctcatc tggtctccct tccgggggac aagacatccc tgtttaatat ttaaacagca
     8461 gtgttcccaa actgggttct tatatccctt gctctggtca accaggttgc agggtttcct
     8521 gtcctcacag gaacgaagtc cctaaagaaa cagtggcagc caggtttagc cccggaattg
     8581 actggattcc ttttttaggg cccattggta tggctttttc cccgtatccc cccaggtgtc
     8641 tgcaggctca aagagcagcg agaagcgttc agaggaaagc gatcccgtgc caccttcccc
     8701 gtgcccgggc tgtccccgca cgctgccggc tcggggatgc ggggggagcg ccggaccgga
     8761 gcggagcccc gggcggctcg ctgctgcccc ctagcggggg agggacgtaa ttacatccct
     8821 gggggctttg ggggggggct gtccctgata tctataacaa gaaaatatat atataataag
     8881 ttatcacgta agtagaacat gaaataacaa tataattatc gtatgagtta aatcttaaaa
     8941 gtcacgtaaa agataatcat gcgtcatttt gactcacgcg gtcgttatag ttcaaaatca
     9001 gtgacactta ccgcattgac aagcacgcct cacgggagct ccaagcggcg actgagatgt
     9061 cctaaatgca cagcgacgga ttcgcgctat ttagaaagag agagcaatat ttcaagaatg
     9121 catgcgtcaa ttttacgcag actatctttc tagggttaat ctagctgcat caggatcata
     9181 tcgtcgggtc ttttttccgg ctcagtcatc gcccaagctg gcgctatctg ggcatcgggg
     9241 aggaagaagc ccgtgccttt tcccgcgagg ttgaagcggc atggaaagag tttgccgagg
     9301 atgactgctg ctgcattgac gttgagcgaa aacgcacgtt taccatgatg attcgggaag
     9361 gtgtggccat gcacgccttt aacggtgaac tgttcgttca ggccacctgg gataccagtt
     9421 cgtcgcggct tttccggaca cagttccgga tggtcagccc gaagcgcatc agcaacccga
     9481 acaataccgg cgacagccgg aactgccgtg ccggtgtgca gattaatgac agcggtgcgg
     9541 cgctgggata ttacgtcagc gaggacgggt atcctggctg gatgccgcag aaatggacat
     9601 ggataccccg tgagttaccc ggcgggcgcg cttggcgtaa tcatggtcat agctgtttcc
     9661 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     9721 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     9781 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     9841 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     9901 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     9961 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
    10021 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
    10081 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
    10141 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
    10201 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
    10261 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
    10321 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
    10381 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
    10441 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
    10501 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
    10561 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
    10621 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
    10681 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
    10741 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
    10801 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
    10861 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
    10921 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
    10981 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
    11041 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
    11101 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
    11161 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
    11221 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
    11281 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
    11341 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
    11401 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
    11461 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
    11521 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
    11581 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
    11641 ggcgacacgg aaatgttgaa tactcat
//
