pORANGE Cloning template vector

LOCUS       pORANGE Cloning template vector              8494 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Cloning template form ORANGE method based knock-in constructs.
ACCESSION   .
VERSION     .
KEYWORDS    pORANGE Cloning template vector
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8494)
  AUTHORS   Willems J, de Jong APH, Scheefhals N, Mertens E, Catsburg LAE, 
            Poorthuis RB, de Winter F, Verhaagen J, Meye FJ, MacGillavry HD
  TITLE     ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope 
            tagging of endogenous proteins in neurons.
  JOURNAL   PLoS Biol. 2020 Apr 10;18(4):e3000665. doi: 
            10.1371/journal.pbio.3000665.
  PUBMED    32275651
REFERENCE   2  (bases 1 to 8494)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..8494
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        62..302
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     62..82
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     233..252
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        329..404
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        559..844
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        846..1123
                     /label=chicken beta-actin promoter
     intron          1124..1351
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1372..1398
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1402..1422
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1432..5532
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5536..5556
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5596..5613)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5602..5809
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5818..5958
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5818..5947
                     /label=AAV2 ITR
     rep_origin      6033..6488
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6120..6139)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6330..6351
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6505..6524)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6624..6646
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6684..6702)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6770..6874
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6875..7735
                     /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(7093..7112)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7906..8494
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8395..8414
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
       61 tgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
      121 gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
      181 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca
      241 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg
      301 acgaaacacc gggtcttcga gaagacctgt tttagagcta gaaatagcaa gttaaaataa
      361 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt gttttagagc
      421 tagaaatagc aagttaaaat aaggctagtc cgtttttagc gcgtgcgcca attctgcaga
      481 caaatggctc tagaagcttg ctagcaattg tcgactcgag gatcctagga cgcgtcctag
      541 gacgcgtcca gaggtacccg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      601 caacgacccc cgcccattga cgtcaatagt aacgccaata gggactttcc attgacgtca
      661 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      721 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt gtgcccagta
      781 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      841 catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc
      901 cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg
      961 gggggggcgc gcgccggggg gggggggggg gggggggggg gggggsgggg sgrggcggag
     1021 aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg
     1081 gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg
     1141 ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac
     1201 cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagct
     1261 gagcaagagg taagggttta agggatggtt ggttggtggg gtattaatgt ttaattacct
     1321 ggagcacctg cctgaaatca ctttttttca ggttggaccg gtgccaccat gtacccatac
     1381 gatgttccag attacgcttc gccgaagaaa aagcgcaagg tcgaagcgtc cgacaagaag
     1441 tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag
     1501 tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag
     1561 aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg
     1621 aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag
     1681 atcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc
     1741 ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac
     1801 gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac
     1861 agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc
     1921 cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg
     1981 ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc
     2041 ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat
     2101 ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg
     2161 agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg
     2221 cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac
     2281 cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac
     2341 atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga
     2401 tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct
     2461 gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac
     2521 ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac
     2581 ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc
     2641 ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg
     2701 cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg
     2761 accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg
     2821 atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag
     2881 ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac
     2941 gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg
     3001 accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag
     3061 aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg
     3121 aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa
     3181 gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag
     3241 gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca
     3301 ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac
     3361 gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg
     3421 aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag
     3481 tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt
     3541 aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt
     3601 gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg
     3661 gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc
     3721 agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc
     3781 gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc
     3841 cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg
     3901 gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag
     3961 agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg
     4021 ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg
     4081 cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag
     4141 agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc
     4201 cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac
     4261 gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc
     4321 gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc
     4381 cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg
     4441 gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag
     4501 agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac
     4561 tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag
     4621 acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg
     4681 aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc
     4741 ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag
     4801 gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg
     4861 gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg
     4921 gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc
     4981 aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc
     5041 gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac
     5101 gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag
     5161 ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac
     5221 tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac
     5281 gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag
     5341 caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc
     5401 aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac
     5461 gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag
     5521 ctgggaggcg acagccccaa gaagaagaga aaggtggagg ccagctaaga attctaacta
     5581 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     5641 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     5701 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     5761 aggacagcaa gggggaggat tgggaagaga atagcaggca tgctggggag cggccgcagg
     5821 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg
     5881 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag
     5941 cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     6001 tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc
     6061 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc
     6121 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     6181 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     6241 acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     6301 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     6361 caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg
     6421 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt
     6481 tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     6541 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     6601 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     6661 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     6721 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     6781 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     6841 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     6901 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     6961 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     7021 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     7081 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     7141 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     7201 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     7261 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     7321 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     7381 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     7441 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     7501 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     7561 tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag cactggggcc
     7621 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     7681 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     7741 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     7801 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     7861 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     7921 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     7981 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     8041 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     8101 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     8161 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     8221 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     8281 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     8341 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     8401 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     8461 gtgatgctcg tcaggggggc ggagcctatg gaaa
//