LOCUS       sequence-275622-        8451 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV and T7 promoter expression plasmid for human codon optimized 
            SpCas9-VRER(D1135V/G1218R/R1335E/T1337R) with a c-terminal 
            bi-partite NLS, 3x flag tag, and P2A-EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8451)
  AUTHORS   Walton RT, Christie KA, Whittaker MN, Kleinstiver BP
  TITLE     Unconstrained genome targeting with near-PAMless engineered 
            CRISPR-Cas9 variants.
  JOURNAL   Science. 2020 Apr 17;368(6488):290-296. doi: 
            10.1126/science.aba8853. Epub 2020 Mar 26.
  PUBMED    32217751
REFERENCE   2  (bases 1 to 8451)
  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: 
            "10.1126/science.aba8853"; journalName: "Science"; date: 
            "2020-04-17- 17"; volume: "368"; issue: "6488"; pages: "290-296"
COMMENT     Sequence Label: pCMV-T7-SpCas9-VRER-P2A-EGFP (RTW3160)
FEATURES             Location/Qualifiers
     source          1..8451
                     /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"
     regulatory      403..412
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(409..3810,3811..3813,3814..4059,4060..4062,4063..4410,
                     4411..4413,4414..4416,4417..4419,4420..4512)
                     /codon_start=1
                     /product="Streptococcus pyogenes Cas9 endonuclease with the
                     D1135V/G1218R/R1335E/T1337R mutations"
                     /label=Cas9 VRER
                     /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
                     ESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             409..4512
                     /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
                     ESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4567..4587
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4600..4665
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4684..4740
                     /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             join(4741..4743,4744..4746,4747..5460)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4741..4743,4744..4746,4747..5457)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5047..5066)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5394..5415
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5466..5483
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5506..5523)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5512..5736
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5807..5823)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5807..5823)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5820..5842)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5831..5847
                     /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(5855..5861,5862..5879,5880..5885))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5900..5921
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6038..6055)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6209..6797)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6289..6308)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6968..7759,7760..7828))
                     /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     7591..7610
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7829..7933)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8012..8031)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(8203..8451,1..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 ggacaagaag
      421 tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag
      481 tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag
      541 aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg
      601 aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag
      661 atcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc
      721 ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac
      781 gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac
      841 agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc
      901 cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg
      961 ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc
     1021 ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat
     1081 ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg
     1141 agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg
     1201 cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac
     1261 cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac
     1321 atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga
     1381 tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct
     1441 gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac
     1501 ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac
     1561 ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc
     1621 ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg
     1681 cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg
     1741 accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg
     1801 atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag
     1861 ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac
     1921 gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg
     1981 accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag
     2041 aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg
     2101 aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa
     2161 gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag
     2221 gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca
     2281 ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac
     2341 gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg
     2401 aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag
     2461 tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt
     2521 aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt
     2581 gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg
     2641 gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc
     2701 agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc
     2761 gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc
     2821 cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg
     2881 gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag
     2941 agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg
     3001 ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg
     3061 cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag
     3121 agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc
     3181 cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac
     3241 gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc
     3301 gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc
     3361 cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg
     3421 gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag
     3481 agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac
     3541 tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag
     3601 acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg
     3661 aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc
     3721 ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag
     3781 gactgggacc ctaagaagta cggcggcttc gtgagcccca ccgtggccta ttctgtgctg
     3841 gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg
     3901 gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc
     3961 aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc
     4021 gagctggaaa acggccggaa gagaatgctg gcctctgcca gagaactgca gaagggaaac
     4081 gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag
     4141 ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac
     4201 tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac
     4261 gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag
     4321 caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc
     4381 aagtactttg acaccaccat cgaccggaag gagtacagaa gcaccaaaga ggtgctggac
     4441 gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag
     4501 ctgggaggcg acggatccgg cggaggcgga agcgggaaaa gaaccgccga cggcagcgaa
     4561 ttcgagccca agaagaagag gaaagtctcg agcggaggcg actacaaaga ccatgacggt
     4621 gattataaag atcatgacat cgattacaag gatgacgatg acaagggagg cggtggaagc
     4681 ggcgcaacaa acttctctct gctgaaacaa gccggagatg tcgaagagaa tcctggaccg
     4741 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     4801 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     4861 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     4921 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     4981 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     5041 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     5101 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     5161 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     5221 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     5281 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     5341 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     5401 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtga
     5461 ccggtcatca tcaccatcac cattgagttt aaacccgctg atcagcctcg actgtgcctt
     5521 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     5581 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     5641 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     5701 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     5761 ggggctcgat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     5821 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     5881 gtaaagccta gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     5941 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     6001 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     6061 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     6121 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     6181 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     6241 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     6301 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     6361 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt
     6421 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     6481 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     6541 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     6601 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     6661 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     6721 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     6781 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     6841 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     6901 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     6961 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     7021 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     7081 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     7141 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     7201 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     7261 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     7321 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     7381 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     7441 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     7501 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     7561 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     7621 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     7681 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     7741 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     7801 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     7861 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     7921 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc
     7981 gatctcccga tcccctaggg tcgactctca gtacaatctg ctctgatgcc gcatagttaa
     8041 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
     8101 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
     8161 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta
     8221 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
     8281 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     8341 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     8401 gggtggacta tttacggtaa actgcccact tggcagtaca tcaagtgtat c
//
