LOCUS       sequence-424421-        8312 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpCas9HF1 and puromycin resistance genes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8312)
  AUTHORS   Qi T, Wang Y, Yang Y, Gao S, Liu J, Huang Q, Tian Y, Tang J, Zheng 
            WV, Wang Y
  TITLE     Phage-assisted evolution of compact Cas9 variants targeting a simple
            NNG PAM.
  JOURNAL   Nat Chem Biol. 2023 Dec 5. doi: 10.1038/s41589-023-01481-5.
  PUBMED    38052959
REFERENCE   2  (bases 1 to 8312)
  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 Chem 
            Biol. 2023 Dec 5. doi: 10.1038/s41589-023-01481-5."
COMMENT     Sequence Label: pCMV-SpCas9HF1-P2A-puro
FEATURES             Location/Qualifiers
     source          1..8312
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        132..335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     286..306
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     377..396
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        377..395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             445..465
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(466..1950,1951..1953,1954..2442,2443..2445,2446..2544,
                     2545..2547,2548..3237,3238..3240,3241..4566)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Kleinstiver et al., 2016)"
                     /label=SpCas9-HF1
                     /note="carries the mutations N497A, R661A, Q695A, and 
                     Q926A"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMA
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             466..4566
                     /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
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMA
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4609..4629
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4639..4695
                     /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             4696..5292
                     /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(4696..4715)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5192..5212
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             5305..5325
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5334..5351
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5374..5391)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5380..5604
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5675..5691)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5675..5691)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5688..5710)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5699..5715
                     /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(5723..5729,5730..5747,5748..5753))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5768..5789
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5899..5916)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6070..6658)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6150..6169)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6829..7620,7621..7689))
                     /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     7452..7471
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7690..7794)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7873..7892)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(8064..8312,1..131)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
       61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gaaacggaca
      421 gccgacggaa gcgagttcga gtcaccaaag aagaagcgga aagtcgacaa gaagtacagc
      481 atcggcctgg acatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag
      541 gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac
      601 ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga
      661 accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc
      721 agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg
      781 gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg
      841 gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc
      901 gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc
      961 cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc
     1021 cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg
     1081 gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc
     1141 gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg
     1201 ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg
     1261 agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac
     1321 gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg
     1381 agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac
     1441 gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag
     1501 tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga
     1561 gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc
     1621 gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac
     1681 aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag
     1741 gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc
     1801 cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc
     1861 agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct
     1921 tccgcccaga gcttcatcga gcggatgacc gccttcgata agaacctgcc caacgagaag
     1981 gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa
     2041 gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag
     2101 gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag
     2161 gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg
     2221 ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc
     2281 ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt
     2341 gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa
     2401 gtgatgaagc agctgaagcg gcggagatac accggctggg gcgccctgag ccggaagctg
     2461 atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac
     2521 ggcttcgcca acagaaactt catggccctg atccacgacg acagcctgac ctttaaagag
     2581 gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat
     2641 ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag
     2701 ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag
     2761 aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag
     2821 ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg
     2881 cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag
     2941 gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc tcagagcttt
     3001 ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag
     3061 agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg
     3121 ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc
     3181 ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga aacccgggcc
     3241 atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat
     3301 gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc
     3361 cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac
     3421 gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc
     3481 gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag
     3541 caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc
     3601 aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac
     3661 ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg
     3721 ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc
     3781 aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg
     3841 gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt gctggtggtg
     3901 gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc
     3961 accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc
     4021 tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg
     4081 gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg aaacgaactg
     4141 gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag
     4201 ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg
     4261 gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat
     4321 ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc
     4381 gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc cttcaagtac
     4441 tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct ggacgccacc
     4501 ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga
     4561 ggcgactctg gcggctcaaa aagaaccgcc gacggcagcg aattcgagcc caagaagaag
     4621 aggaaagtcg gaagcggagc tactaacttc agcctgctga agcaggctgg agacgtggag
     4681 gagaaccctg gacctatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac
     4741 gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac
     4801 accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg
     4861 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc
     4921 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg
     4981 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg
     5041 caccggccca aggagcccgc gtggttcctg gccaccgtcg gagtctcgcc cgaccaccag
     5101 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg
     5161 gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc
     5221 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc
     5281 aagcccggtg cctctggtgg ttctcccaag aagaaaagga aagtctaacc ggtcatcatc
     5341 accatcacca ttgagtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc
     5401 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     5461 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     5521 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     5581 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctcgatac
     5641 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     5701 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctagg
     5761 atgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     5821 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcggga agaggcggtt
     5881 tgcgtattgg gccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     5941 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     6001 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     6061 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     6121 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     6181 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     6241 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     6301 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     6361 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     6421 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     6481 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     6541 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     6601 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     6661 agatcctttg atcttttcta cggggtctga cactcagtgg aacgaaaact cacgttaagg
     6721 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     6781 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     6841 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     6901 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     6961 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     7021 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     7081 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     7141 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     7201 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     7261 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     7321 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     7381 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     7441 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     7501 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     7561 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     7621 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     7681 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     7741 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     7801 tccccgaaaa gtgccacctg acgtcgacgg atcgggagat cgatctcccg atcccctagg
     7861 gtcgactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc
     7921 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg
     7981 cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga
     8041 tgtacgggcc agatatacgc gttgacattg attattgact agttattaat agtaatcaat
     8101 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
     8161 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
     8221 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     8281 aactgcccac ttggcagtac atcaagtgta tc
//
