LOCUS       sequence-373493-       12824 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FLAG tagged SpCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12824)
  AUTHORS   Escobar M, Li J, Patel A, Liu S, Xu Q, Hilton IB
  TITLE     Quantification of Genome Editing and Transcriptional Control 
            Capabilities Reveals Hierarchies among Diverse CRISPR/Cas Systems in
            Human Cells.
  JOURNAL   ACS Synth Biol. 2022 Oct 21;11(10):3239-3250. doi: 
            10.1021/acssynbio.2c00156. Epub 2022 Sep 26.
  PUBMED    36162812
REFERENCE   2  (bases 1 to 12824)
  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: 
            "10.1021/acssynbio.2c00156"; journalName: "ACS Synth Biol"; date: 
            "2022-10-21- 21"; volume: "11"; issue: "10"; pages: "3239-3250"
COMMENT     Sequence Label: SpCas9
FEATURES             Location/Qualifiers
     source          1..12824
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..199
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     154..174
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             217..397
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    444..569
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1062..1295
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1480..1524
                     /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    1822..1939
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_RNA        2071..2146
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2208..2419
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2438..2447
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2444..6547
                     /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             2444..6547
                     /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             6548..6595
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6596..6619
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6629..6685
                     /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             6686..7282
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     7179..7199
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7298..7886
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7351..7371)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7769..7780)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             7958..8191
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(8217..8234)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8223..8447
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8493..8921
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8580..8599)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8790..8811
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8930..8950)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        8935..9264
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9115..9250
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9177..9196
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9312..9359
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9378..9752
                     /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    9882..10003
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9919..9938)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9973..9992
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10052..10068)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10052..10068)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10065..10087)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10076..10092
                     /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(10100..10106,10107..10124,10125..10130))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10145..10166
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10283..10300)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10454..11042)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10534..10553)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11213..12004,12005..12073))
                     /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     11836..11855
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12074..12178)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12253..12272)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        12444..12823
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
       61 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
      121 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
      181 gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc
      241 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
      301 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
      361 ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga
      421 aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca
      481 cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct
      541 agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat
      601 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
      661 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
      721 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
      781 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
      841 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac
      901 agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
      961 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
     1021 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
     1081 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
     1141 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
     1201 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
     1261 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
     1321 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
     1381 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
     1441 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg
     1501 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat
     1561 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta
     1621 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc
     1681 ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac
     1741 agagacagat ccattcgatt agtgaacgga tcggcactgc gtgcgccaat tctgcagaca
     1801 aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg
     1861 ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat
     1921 tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag tttggttaat
     1981 taaggtaccg atatcactag tgttccatta actgcgctaa aaataatttt taaatctttt
     2041 ttagcttctt gctctttttt gtacgtctct gttttagagc tagaaatagc aagttaaaat
     2101 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgaattcgc
     2161 tagctaggtc ttgaaaggag tgggaattgg ctccggtgcc cgtcagtggg cagagcgcac
     2221 atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgatccg gtgcctagag
     2281 aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga
     2341 gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg
     2401 gtttgccgcc agaacacagg accggttcta gagcgctgcc accatggaca agaagtacag
     2461 catcggcctg gacatcggca ccaactctgt gggctgggcc gtgatcaccg acgagtacaa
     2521 ggtgcccagc aagaaattca aggtgctggg caacaccgac cggcacagca tcaagaagaa
     2581 cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag
     2641 aaccgccaga agaagataca ccagacggaa gaaccggatc tgctatctgc aagagatctt
     2701 cagcaacgag atggccaagg tggacgacag cttcttccac agactggaag agtccttcct
     2761 ggtggaagag gataagaagc acgagcggca ccccatcttc ggcaacatcg tggacgaggt
     2821 ggcctaccac gagaagtacc ccaccatcta ccacctgaga aagaaactgg tggacagcac
     2881 cgacaaggcc gacctgcggc tgatctatct ggccctggcc cacatgatca agttccgggg
     2941 ccacttcctg atcgagggcg acctgaaccc cgacaacagc gacgtggaca agctgttcat
     3001 ccagctggtg cagacctaca accagctgtt cgaggaaaac cccatcaacg ccagcggcgt
     3061 ggacgccaag gccatcctgt ctgccagact gagcaagagc agacggctgg aaaatctgat
     3121 cgcccagctg cccggcgaga agaagaatgg cctgttcgga aacctgattg ccctgagcct
     3181 gggcctgacc cccaacttca agagcaactt cgacctggcc gaggatgcca aactgcagct
     3241 gagcaaggac acctacgacg acgacctgga caacctgctg gcccagatcg gcgaccagta
     3301 cgccgacctg tttctggccg ccaagaacct gtccgacgcc atcctgctga gcgacatcct
     3361 gagagtgaac accgagatca ccaaggcccc cctgagcgcc tctatgatca agagatacga
     3421 cgagcaccac caggacctga ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa
     3481 gtacaaagag attttcttcg accagagcaa gaacggctac gccggctaca ttgacggcgg
     3541 agccagccag gaagagttct acaagttcat caagcccatc ctggaaaaga tggacggcac
     3601 cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc ggaccttcga
     3661 caacggcagc atcccccacc agatccacct gggagagctg cacgccattc tgcggcggca
     3721 ggaagatttt tacccattcc tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt
     3781 ccgcatcccc tactacgtgg gccctctggc caggggaaac agcagattcg cctggatgac
     3841 cagaaagagc gaggaaacca tcaccccctg gaacttcgag gaagtggtgg acaagggcgc
     3901 ttccgcccag agcttcatcg agcggatgac caacttcgat aagaacctgc ccaacgagaa
     3961 ggtgctgccc aagcacagcc tgctgtacga gtacttcacc gtgtataacg agctgaccaa
     4021 agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa
     4081 ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga
     4141 ggactacttc aagaaaatcg agtgcttcga ctccgtggaa atctccggcg tggaagatcg
     4201 gttcaacgcc tccctgggca cataccacga tctgctgaaa attatcaagg acaaggactt
     4261 cctggacaat gaggaaaacg aggacattct ggaagatatc gtgctgaccc tgacactgtt
     4321 tgaggacaga gagatgatcg aggaacggct gaaaacctat gcccacctgt tcgacgacaa
     4381 agtgatgaag cagctgaagc ggcggagata caccggctgg ggcaggctga gccggaagct
     4441 gatcaacggc atccgggaca agcagtccgg caagacaatc ctggatttcc tgaagtccga
     4501 cggcttcgcc aacagaaact tcatgcagct gatccacgac gacagcctga cctttaaaga
     4561 ggacatccag aaagcccagg tgtccggcca gggcgatagc ctgcacgagc acattgccaa
     4621 tctggccggc agccccgcca ttaagaaggg catcctgcag acagtgaagg tggtggacga
     4681 gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga
     4741 gaaccagacc acccagaagg gacagaagaa cagccgcgag agaatgaagc ggatcgaaga
     4801 gggcatcaaa gagctgggca gccagatcct gaaagaacac cccgtggaaa acacccagct
     4861 gcagaacgag aagctgtacc tgtactacct gcagaatggg cgggatatgt acgtggacca
     4921 ggaactggac atcaaccggc tgtccgacta cgatgtggac catatcgtgc ctcagagctt
     4981 tctgaaggac gactccatcg acaacaaggt gctgaccaga agcgacaaga accggggcaa
     5041 gagcgacaac gtgccctccg aagaggtcgt gaagaagatg aagaactact ggcggcagct
     5101 gctgaacgcc aagctgatta cccagagaaa gttcgacaat ctgaccaagg ccgagagagg
     5161 cggcctgagc gaactggata aggccggctt catcaagaga cagctggtgg aaacccggca
     5221 gatcacaaag cacgtggcac agatcctgga ctcccggatg aacactaagt acgacgagaa
     5281 tgacaagctg atccgggaag tgaaagtgat caccctgaag tccaagctgg tgtccgattt
     5341 ccggaaggat ttccagtttt acaaagtgcg cgagatcaac aactaccacc acgcccacga
     5401 cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta agctggaaag
     5461 cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga
     5521 gcaggaaatc ggcaaggcta ccgccaagta cttcttctac agcaacatca tgaacttttt
     5581 caagaccgag attaccctgg ccaacggcga gatccggaag cggcctctga tcgagacaaa
     5641 cggcgaaacc ggggagatcg tgtgggataa gggccgggat tttgccaccg tgcggaaagt
     5701 gctgagcatg ccccaagtga atatcgtgaa aaagaccgag gtgcagacag gcggcttcag
     5761 caaagagtct atcctgccca agaggaacag cgataagctg atcgccagaa agaaggactg
     5821 ggaccctaag aagtacggcg gcttcgacag ccccaccgtg gcctattctg tgctggtggt
     5881 ggccaaagtg gaaaagggca agtccaagaa actgaagagt gtgaaagagc tgctggggat
     5941 caccatcatg gaaagaagca gcttcgagaa gaatcccatc gactttctgg aagccaaggg
     6001 ctacaaagaa gtgaaaaagg acctgatcat caagctgcct aagtactccc tgttcgagct
     6061 ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact
     6121 ggccctgccc tccaaatatg tgaacttcct gtacctggcc agccactatg agaagctgaa
     6181 gggctccccc gaggataatg agcagaaaca gctgtttgtg gaacagcaca agcactacct
     6241 ggacgagatc atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa
     6301 tctggacaaa gtgctgtccg cctacaacaa gcaccgggat aagcccatca gagagcaggc
     6361 cgagaatatc atccacctgt ttaccctgac caatctggga gcccctgccg ccttcaagta
     6421 ctttgacacc accatcgacc ggaagaggta caccagcacc aaagaggtgc tggacgccac
     6481 cctgatccac cagagcatca ccggcctgta cgagacacgg atcgacctgt ctcagctggg
     6541 aggcgacaag cgacctgccg ccacaaagaa ggctggacag gctaagaaga agaaagatta
     6601 caaagacgat gacgataagg gatccggcgc aacaaacttc tctctgctga aacaagccgg
     6661 agatgtcgaa gagaatcctg gaccgaccga gtacaagccc acggtgcgcc tcgccacccg
     6721 cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac
     6781 gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt
     6841 cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt
     6901 ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc
     6961 gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct
     7021 ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggag tctcgcccga
     7081 ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg
     7141 cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg
     7201 gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat
     7261 gacccgcaag cccggtgcct gaacgcgtta agtcgacaat caacctctgg attacaaaat
     7321 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     7381 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     7441 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     7501 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     7561 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     7621 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     7681 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct
     7741 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     7801 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     7861 gatctccctt tgggccgcct ccccgcgtcg actttaagac caatgactta caaggcagct
     7921 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa
     7981 cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc
     8041 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
     8101 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga
     8161 cccttttagt cagtgtggaa aatctctagc agggcccgtt taaacccgct gatcagcctc
     8221 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     8281 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     8341 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     8401 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     8461 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     8521 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     8581 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     8641 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     8701 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     8761 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     8821 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     8881 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     8941 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     9001 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     9061 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     9121 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     9181 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     9241 tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct
     9301 gatcagcacg tgttgacaat taatcatcgg catagtatat cggcatagta taatacgaca
     9361 aggtgaggaa ctaaaccatg gccaagttga ccagtgccgt tccggtgctc accgcgcgcg
     9421 acgtcgccgg agcggtcgag ttctggaccg accggctcgg gttctcccgg gacttcgtgg
     9481 aggacgactt cgccggtgtg gtccgggacg acgtgaccct gttcatcagc gcggtccagg
     9541 accaggtggt gccggacaac accctggcct gggtgtgggt gcgcggcctg gacgagctgt
     9601 acgccgagtg gtcggaggtc gtgtccacga acttccggga cgcctccggg ccggccatga
     9661 ccgagatcgg cgagcagccg tgggggcggg agttcgccct gcgcgacccg gccggcaact
     9721 gcgtgcactt cgtggccgag gagcaggact gacacgtgct acgagatttc gattccaccg
     9781 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     9841 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     9901 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     9961 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
    10021 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
    10081 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
    10141 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
    10201 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
    10261 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
    10321 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
    10381 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
    10441 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
    10501 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
    10561 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
    10621 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
    10681 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
    10741 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
    10801 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
    10861 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
    10921 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
    10981 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
    11041 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
    11101 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
    11161 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
    11221 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
    11281 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
    11341 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
    11401 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
    11461 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
    11521 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
    11581 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
    11641 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
    11701 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
    11761 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
    11821 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
    11881 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
    11941 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
    12001 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
    12061 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
    12121 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
    12181 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg
    12241 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc
    12301 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg
    12361 cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga
    12421 tgtacgggcc agatatacgc gttgacattg attattgact agttattaat agtaatcaat
    12481 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
    12541 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
    12601 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
    12661 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
    12721 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
    12781 tacttggcag tacatctacg tattagtcat cgctattacc atgg
//
