AIO-GFP

LOCUS       AIO-GFP                9740 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  All-in-One plasmid encoding dual U6 promoter-driven sgRNAs and 
            EGFP-coupled Cas9-D10A nickase to enhance efficient and accurate 
            genome editing.
ACCESSION   .
VERSION     .
KEYWORDS    AIO-GFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9740)
  AUTHORS   Chiang TW, le Sage C, Larrieu D, Demir M, Jackson SP
  TITLE     CRISPR-Cas9(D10A) nickase-based genotypic and phenotypic screening 
            to enhance genome editing.
  JOURNAL   Sci Rep. 2016 Apr 15;6:24356. doi: 10.1038/srep24356.
  PUBMED    27079678
REFERENCE   2  (bases 1 to 9740)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..9740
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..241
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        277..352
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        442..459
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        477..717
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        752..827
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(836..854)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(837..854)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        891..1176
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        1178..1455
                     /label=chicken beta-actin promoter
     intron          1456..1684
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1696..1705
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1705..1770
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1777..1797
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1822..5922
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5923..5970
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5986..6039
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             6040..6753
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             6040..6753
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(6082..6103)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6343..6362)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6690..6711
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6781..6798)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6787..6994
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7003..7143
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7003..7132
                     /label=AAV2 ITR
     rep_origin      7218..7673
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7305..7324)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7515..7536
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7690..7709)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7809..7831
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7869..7887)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7955..8059
                     /gene="bla"
                     /label=AmpR promoter
     CDS             8060..8920
                     /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     complement(8278..8297)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9091..9679
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9580..9599
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg gggtcttcgg atccatgcga agacctgttt tagagctaga aatagcaagt
      301 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttgt
      361 tttagagcta gaaatagcaa gttaaaataa ggctagtccg tttttagcgc gtgcgccaat
      421 tctgcagaca aatggctcta gattaaccct cactaaaggg aaaggtcggg caggaagagg
      481 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
      541 ttagaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
      601 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
      661 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa
      721 acaccgggag accatcgatg agagggtctc agttttagag ctagaaatag caagttaaaa
      781 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttttctat
      841 agtgtcacct aaattgcagc tctggcccgt gtctcaaaat ctctggtacc cgttacataa
      901 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
      961 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     1021 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
     1081 ggtaaatggc ccgcctggca ttgtgcccag tacatgacct tatgggactt tcctacttgg
     1141 cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc
     1201 ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa
     1261 ttattttgtg cagcgatggg ggcggggggg gggggggggc gcgcgccagg cggggcgggg
     1321 cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc
     1381 gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga
     1441 agcgcgcggc gggcgggagt cgctgcgacg ctgccttcgc cccgtgcccc gctccgccgc
     1501 cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg
     1561 ggacggccct tctcctccgg gctgtaatta gctgagcaag aggtaagggt ttaagggatg
     1621 gttggttggt ggggtattaa tgtttaatta cctggagcac ctgcctgaaa tcactttttt
     1681 tcaggttgga ccggtgccac catggactat aaggaccacg acggagacta caaggatcat
     1741 gatattgatt acaaagacga tgacgataag atggccccaa agaagaagcg gaaggtcggt
     1801 atccacggag tcccagcagc cgacaagaag tacagcatcg gcctggccat cggcaccaac
     1861 tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg
     1921 ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct gctgttcgac
     1981 agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag atacaccaga
     2041 cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac
     2101 gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag
     2161 cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc
     2221 atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc
     2281 tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg
     2341 aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag
     2401 ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc
     2461 agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag
     2521 aatggcctgt tcggcaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc
     2581 aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac
     2641 ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag
     2701 aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag
     2761 gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg
     2821 ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag
     2881 agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga gttctacaag
     2941 ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac
     3001 agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc
     3061 cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag
     3121 gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct
     3181 ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc
     3241 ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt catcgagcgg
     3301 atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg
     3361 tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac cgagggaatg
     3421 agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct gctgttcaag
     3481 accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc
     3541 ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac
     3601 cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac
     3661 attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa
     3721 cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg
     3781 agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag
     3841 tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg
     3901 cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc
     3961 ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag
     4021 aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac
     4081 aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag
     4141 aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag
     4201 atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac
     4261 tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc
     4321 gactacgatg tggaccatat cgtgcctcag agctttctga aggacgactc catcgacaac
     4381 aaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag
     4441 gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag
     4501 agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc
     4561 ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc
     4621 ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg ggaagtgaaa
     4681 gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa
     4741 gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga
     4801 accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag
     4861 gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc
     4921 aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac
     4981 ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg
     5041 gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc
     5101 gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg
     5161 aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc
     5221 gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc
     5281 aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc
     5341 gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg
     5401 atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg
     5461 gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac
     5521 ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag
     5581 aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc
     5641 gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac
     5701 aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc
     5761 ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag
     5821 aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc
     5881 ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acaaaaggcc ggcggccacg
     5941 aaaaaggccg gccaggcaaa aaagaaaaag gaattcggca gtggagaggg cagaggaagt
     6001 ctgctaacat gcggtgacgt cgaggagaat cctggcccag tgagcaaggg cgaggagctg
     6061 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     6121 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     6181 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     6241 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     6301 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     6361 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     6421 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     6481 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     6541 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     6601 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg
     6661 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     6721 gggatcactc tcggcatgga cgagctgtac aaggaattct aactagagct cgctgatcag
     6781 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     6841 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     6901 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     6961 aggattggga agagaatagc aggcatgctg gggagcggcc gcaggaaccc ctagtgatgg
     7021 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg
     7081 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc
     7141 aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     7201 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     7261 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     7321 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     7381 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     7441 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     7501 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggg
     7561 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     7621 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
     7681 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     7741 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     7801 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     7861 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     7921 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     7981 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     8041 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     8101 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     8161 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     8221 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     8281 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     8341 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     8401 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     8461 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     8521 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     8581 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     8641 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     8701 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     8761 agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     8821 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     8881 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     8941 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     9001 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     9061 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     9121 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     9181 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     9241 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     9301 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     9361 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     9421 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     9481 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     9541 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     9601 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     9661 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     9721 ctggcctttt gctcacatgt
//