LOCUS       sequence-447697-        9591 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pCMV and pT7 Human expression plasmid for TadCBEd using SpCas9 CBE 
            enzyme with MKRCKS 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(MKRCKS)-P2A-EGFP (RAS3445)
FEATURES             Location/Qualifiers
     source          1..9591
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        123..502
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        503..706
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     657..677
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     748..767
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        748..766
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             816..836
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1431..5531
                     /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
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKKYSSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5562..5810
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5841..6089
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             6132..6152
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6162..6218
                     /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(6219..6221,6222..6224,6225..6935)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(6219..6221,6222..6224,6225..6935)
                     /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(6525..6544)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6872..6893
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             6948..6968
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6977..6994
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(7017..7034)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7023..7247
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(7318..7334)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7318..7334)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7331..7353)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7342..7358
                     /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(7366..7372,7373..7390,7391..7396))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7411..7432
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7549..7566)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7720..8308)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7800..7819)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8479..9270,9271..9339))
                     /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     9102..9121
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9340..9444)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(9523..9542)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
       61 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
      121 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
      181 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      241 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      301 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      361 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      421 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      481 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
      541 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
      601 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
      661 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaaccg
      721 tcagatccgc tagagatccg cggccgctaa tacgactcac tatagggaga gccgccacca
      781 tgaaacggac agccgacgga agcgagttcg agtcaccaaa gaagaagcgg aaagtctctg
      841 aggtggagtt ttcccacgag tactggatga gacatgccct gaccctggcc aagagggcac
      901 gcgatgagag gaaggcccct gtgggagccg tgctggtgct gaacaataga gtgatcggcg
      961 agggctggaa cagagccatc ggcctgcacg acccaacagc ccatgccgaa attatcgccc
     1021 tgagacaggg cggcctggtc atgcagaact acagactgat tgacgccacc ctgtacgtga
     1081 cattcgagcc ttgcgtgatg tgcgccggcg ccatgatcaa ctctaggatc ggccgcgtgg
     1141 tgtttggcgt gaggaacagc aaaagaggcg ccgcaggctc cctgatgaac gtgctgaact
     1201 accccggcat gaatcaccgc gtcgaaatta ccgagggaat cctggcagat gaatgtgccg
     1261 ccctgctgtg cgatttctac cggatgccta gacaggtgtt caatgctcag aagaaggccc
     1321 agagctccat caactccgga ggatctagcg gaggctcctc tggctctgag acacctggca
     1381 caagcgagag cgcaacacct gaaagcagcg ggggcagcag cggggggtca gacaagaagt
     1441 acagcatcgg cctggccatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt
     1501 acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga
     1561 agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga
     1621 agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga
     1681 tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct
     1741 tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg
     1801 aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca
     1861 gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc
     1921 ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt
     1981 tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg
     2041 gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc
     2101 tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga
     2161 gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc
     2221 agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc
     2281 agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca
     2341 tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat
     2401 acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg
     2461 agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg
     2521 gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg
     2581 gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct
     2641 tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc
     2701 ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga
     2761 ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga
     2821 tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg
     2881 gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg
     2941 agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga
     3001 ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga
     3061 aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga
     3121 aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag
     3181 atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg
     3241 acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac
     3301 tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg
     3361 acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga
     3421 agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt
     3481 ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta
     3541 aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg
     3601 ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg
     3661 acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca
     3721 gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg
     3781 aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc
     3841 agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg
     3901 accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga
     3961 gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg
     4021 gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc
     4081 agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga
     4141 gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc
     4201 ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg
     4261 agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg
     4321 atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc
     4381 acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg
     4441 aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga
     4501 gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact
     4561 ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga
     4621 caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga
     4681 aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct
     4741 tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg
     4801 actgggaccc taagaagtac ggcggcttca tgaagcccac cgtggcctat tctgtgctgg
     4861 tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg
     4921 ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca
     4981 agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg
     5041 agctggaaaa cggccggaag agaatgctgg cctctgccag atgcctgcag aagggaaacg
     5101 aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc
     5161 tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact
     5221 acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg
     5281 ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc
     5341 aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca
     5401 agtactttga caccaccatc gaccggaaga agtacagcag caccaaagag gtgctggacg
     5461 ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc
     5521 tgggaggtga cagcggcggg agcggcggga gcggggggag cactaatctg agcgacatca
     5581 ttgagaagga gactgggaaa cagctggtca ttcaggagtc catcctgatg ctgcctgagg
     5641 aggtggagga agtgatcggc aacaagccag agtctgacat cctggtgcac accgcctacg
     5701 acgagtccac agatgagaat gtgatgctgc tgacctctga cgcccccgag tataagcctt
     5761 gggccctggt catccaggat tctaacggcg agaataagat caagatgctg agcggaggat
     5821 ccggaggatc tggaggcagc accaacctgt ctgacatcat cgagaaggag acaggcaagc
     5881 agctggtcat ccaggagagc atcctgatgc tgcccgaaga agtcgaagaa gtgatcggaa
     5941 acaagcctga gagcgatatc ctggtccata ccgcctacga cgagagtacc gacgaaaatg
     6001 tgatgctgct gacatccgac gccccagagt ataagccctg ggctctggtc atccaggatt
     6061 ccaacggaga gaacaaaatc aaaatgctgt ctggcggctc aaaaagaacc gccgacggca
     6121 gcgaattcga gcccaagaag aagaggaaag tcggaagcgg agctactaac ttcagcctgc
     6181 tgaagcaggc tggagacgtg gaggagaacc ctggacctat ggtgagcaag ggcgaggagc
     6241 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     6301 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     6361 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctatg
     6421 gagtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     6481 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     6541 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     6601 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     6661 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
     6721 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     6781 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc
     6841 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     6901 ccgggatcac tctcggcatg gacgagctgt acaagtctgg tggttctccc aagaagaaga
     6961 ggaaagtcta accggtcatc atcaccatca ccattgagtt taaacccgct gatcagcctc
     7021 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     7081 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     7141 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     7201 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     7261 aagaaccagc tggggctcga taccgtcgac ctctagctag agcttggcgt aatcatggtc
     7321 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     7381 aagcataaag tgtaaagcct agggtgccta atgagtgagc taactcacat taattgcgtt
     7441 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     7501 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     7561 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     7621 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     7681 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     7741 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     7801 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     7861 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     7921 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     7981 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     8041 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     8101 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     8161 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     8221 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     8281 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     8341 cactcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     8401 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     8461 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     8521 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     8581 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     8641 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     8701 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     8761 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     8821 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     8881 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     8941 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     9001 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     9061 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     9121 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     9181 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     9241 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     9301 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     9361 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     9421 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg
     9481 atcgggagat cgatctcccg atcccctagg gtcgactctc agtacaatct gctctgatgc
     9541 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg a
//
