LOCUS       sequence-447698-        9591 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pCMV and pT7 Human expression plasmid for TadCBEd using SpCas9 CBE 
            enzyme with MKRCMV amino acids at positions 
            1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9591)
  AUTHORS   Silverstein RA, Kim N, Kroell AS, Walton RT, Delano J, Butcher RM, 
            Pacesa M, Smith BK, Christie KA, Ha LL, Meis RJ, Clark AB, Spinner 
            AD, Lazzarotto CR, Li Y, Matsubara A, Urbina EO, Dahl GA, Correia 
            BE, Marks DS, Tsai SQ, Pinello L, De Ravin SS, Liu Q, Kleinstiver BP
  TITLE     Custom CRISPR-Cas9 PAM variants via scalable engineering and machine
            learning.
  JOURNAL   Nature. 2025 Apr 22. doi: 10.1038/s41586-025-09021-y.
  PUBMED    40262634
REFERENCE   2  (bases 1 to 9591)
  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: "Nature. 
            2025 Apr 22. doi: 10.1038/s41586-025-09021-y."
COMMENT     Sequence Label: pCMV-T7-TadCBEd-SpCas9(MKRCMV)-P2A-EGFP (BKS1032)
FEATURES             Location/Qualifiers
     source          1..9591
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        133..512
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        513..716
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     667..687
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     758..777
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        758..776
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             826..846
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1441..5541
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFMKPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARCLQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKMYVSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5572..5820
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5851..6099
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             6142..6162
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6172..6228
                     /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(6229..6231,6232..6234,6235..6945)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(6229..6231,6232..6234,6235..6945)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6535..6554)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6882..6903
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             6958..6978
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6987..7004
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(7027..7044)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7033..7257
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(7328..7344)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7328..7344)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7341..7363)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7352..7368
                     /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(7376..7382,7383..7400,7401..7406))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7421..7442
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7559..7576)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7730..8318)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7810..7829)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8489..9280,9281..9349))
                     /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     9112..9131
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9350..9454)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(9533..9552)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
       61 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
      121 gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
      181 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
      241 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
      301 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
      361 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
      421 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
      481 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
      541 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
      601 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
      661 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctggtt
      721 tagtgaaccg tcagatccgc tagagatccg cggccgctaa tacgactcac tatagggaga
      781 gccgccacca tgaaacggac agccgacgga agcgagttcg agtcaccaaa gaagaagcgg
      841 aaagtctctg aggtggagtt ttcccacgag tactggatga gacatgccct gaccctggcc
      901 aagagggcac gcgatgagag gaaggcccct gtgggagccg tgctggtgct gaacaataga
      961 gtgatcggcg agggctggaa cagagccatc ggcctgcacg acccaacagc ccatgccgaa
     1021 attatcgccc tgagacaggg cggcctggtc atgcagaact acagactgat tgacgccacc
     1081 ctgtacgtga cattcgagcc ttgcgtgatg tgcgccggcg ccatgatcaa ctctaggatc
     1141 ggccgcgtgg tgtttggcgt gaggaacagc aaaagaggcg ccgcaggctc cctgatgaac
     1201 gtgctgaact accccggcat gaatcaccgc gtcgaaatta ccgagggaat cctggcagat
     1261 gaatgtgccg ccctgctgtg cgatttctac cggatgccta gacaggtgtt caatgctcag
     1321 aagaaggccc agagctccat caactccgga ggatctagcg gaggctcctc tggctctgag
     1381 acacctggca caagcgagag cgcaacacct gaaagcagcg ggggcagcag cggggggtca
     1441 gacaagaagt acagcatcgg cctggccatc ggcaccaact ctgtgggctg ggccgtgatc
     1501 accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac
     1561 agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc
     1621 acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat
     1681 ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg
     1741 gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac
     1801 atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa
     1861 ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg
     1921 atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg
     1981 gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc
     2041 aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg
     2101 ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg
     2161 attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat
     2221 gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag
     2281 atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg
     2341 ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg
     2401 atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag
     2461 cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc
     2521 tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa
     2581 aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag
     2641 cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc
     2701 attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag
     2761 aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga
     2821 ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg
     2881 gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac
     2941 ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat
     3001 aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc
     3061 ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg
     3121 aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc
     3181 ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc
     3241 aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg
     3301 accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac
     3361 ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg
     3421 ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat
     3481 ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc
     3541 ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac
     3601 gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg
     3661 aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc
     3721 gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg
     3781 aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg
     3841 gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat
     3901 atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc
     3961 gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac
     4021 aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac
     4081 tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc
     4141 aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg
     4201 gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact
     4261 aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag
     4321 ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac
     4381 caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac
     4441 cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg
     4501 atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac
     4561 atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct
     4621 ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc
     4681 accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag
     4741 acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc
     4801 agaaagaagg actgggaccc taagaagtac ggcggcttca tgaagcccac cgtggcctat
     4861 tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa
     4921 gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt
     4981 ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac
     5041 tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccag atgcctgcag
     5101 aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac
     5161 tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag
     5221 cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc
     5281 ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc
     5341 atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct
     5401 gccgccttca agtactttga caccaccatc gaccggaaga tgtacgtgag caccaaagag
     5461 gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac
     5521 ctgtctcagc tgggaggtga cagcggcggg agcggcggga gcggggggag cactaatctg
     5581 agcgacatca ttgagaagga gactgggaaa cagctggtca ttcaggagtc catcctgatg
     5641 ctgcctgagg aggtggagga agtgatcggc aacaagccag agtctgacat cctggtgcac
     5701 accgcctacg acgagtccac agatgagaat gtgatgctgc tgacctctga cgcccccgag
     5761 tataagcctt gggccctggt catccaggat tctaacggcg agaataagat caagatgctg
     5821 agcggaggat ccggaggatc tggaggcagc accaacctgt ctgacatcat cgagaaggag
     5881 acaggcaagc agctggtcat ccaggagagc atcctgatgc tgcccgaaga agtcgaagaa
     5941 gtgatcggaa acaagcctga gagcgatatc ctggtccata ccgcctacga cgagagtacc
     6001 gacgaaaatg tgatgctgct gacatccgac gccccagagt ataagccctg ggctctggtc
     6061 atccaggatt ccaacggaga gaacaaaatc aaaatgctgt ctggcggctc aaaaagaacc
     6121 gccgacggca gcgaattcga gcccaagaag aagaggaaag tcggaagcgg agctactaac
     6181 ttcagcctgc tgaagcaggc tggagacgtg gaggagaacc ctggacctat ggtgagcaag
     6241 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     6301 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     6361 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     6421 ctgacctatg gagtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     6481 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     6541 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     6601 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     6661 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     6721 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     6781 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     6841 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     6901 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtctgg tggttctccc
     6961 aagaagaaga ggaaagtcta accggtcatc atcaccatca ccattgagtt taaacccgct
     7021 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     7081 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     7141 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     7201 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     7261 ctgaggcgga aagaaccagc tggggctcga taccgtcgac ctctagctag agcttggcgt
     7321 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     7381 tacgagccgg aagcataaag tgtaaagcct agggtgccta atgagtgagc taactcacat
     7441 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     7501 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     7561 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     7621 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     7681 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     7741 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     7801 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     7861 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     7921 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     7981 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     8041 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     8101 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     8161 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     8221 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     8281 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     8341 cggggtctga cactcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     8401 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     8461 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     8521 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     8581 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     8641 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     8701 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     8761 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     8821 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     8881 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     8941 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     9001 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     9061 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     9121 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     9181 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     9241 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     9301 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     9361 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     9421 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     9481 acgtcgacgg atcgggagat cgatctcccg atcccctagg gtcgactctc agtacaatct
     9541 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg g
//
