LOCUS       sequence-236350-        9034 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The plasmids pSpCas9 (BB)-2A-Puro (Addgene, #48139) and eSpCas9 
            (1.1) (Addgene, #71814) were combined to express both "enhanced 
            specificity" Cas9 and puromycin resistance protein. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9034)
  AUTHORS   Liu Y, Dakou E, Meng Y, Leyns L
  TITLE     Loss of Emp2 compromises cardiogenic differentiation in mouse 
            embryonic stem cells.
  JOURNAL   Biochem Biophys Res Commun. 2019 Feb 14. pii: S0006-291X(19)30235-9.
            doi: 10.1016/j.bbrc.2019.02.048.
  PUBMED    30773261
REFERENCE   2  (bases 1 to 9034)
  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: "Biochem 
            Biophys Res Commun. 2019 Feb 14. pii: S0006-291X(19)30235-9. doi: 
            10.1016/j.bbrc.2019.02.048."
COMMENT     Sequence Label: eSpCas9(1.1)-Puro
FEATURES             Location/Qualifiers
     source          1..9034
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          27..254
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      266..275
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             275..340
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             347..367
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(392..2929,2930..2932,2933..3394,3395..3397,3398..3565,
                     3566..3568,3569..4492)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Slaymaker et al., 2016)"
                     /label=eSpCas9(1.1)
                     /note="carries the mutations K848A, K1003A, and R1060A"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4493..4540
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4556..4609
                     /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             4610..5209
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APHNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4610..4629)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     complement(5237..5254)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5243..5450
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5459..5599
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5459..5588
                     /label=AAV2 ITR
     rep_origin      5674..6129
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5761..5780)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5971..5992
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6146..6165)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6265..6287
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6325..6343)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6411..6515
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6516..6584,6585..7376)
                     /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(6734..6753)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7547..8135
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8036..8055
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8197..8445
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8197..8217
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8368..8387
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8464..8539
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        8636..8921
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc
       61 gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg
      121 tgagcgggcg ggacggccct tctcctccgg gctgtaatta gctgagcaag aggtaagggt
      181 ttaagggatg gttggttggt ggggtattaa tgtttaatta cctggagcac ctgcctgaaa
      241 tcactttttt tcaggttgga ccggtgccac catggactat aaggaccacg acggagacta
      301 caaggatcat gatattgatt acaaagacga tgacgataag atggccccaa agaagaagcg
      361 gaaggtcggt atccacggag tcccagcagc cgacaagaag tacagcatcg gcctggacat
      421 cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa
      481 attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct
      541 gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag
      601 atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc
      661 caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa
      721 gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa
      781 gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct
      841 gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga
      901 gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac
      961 ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat
     1021 cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg
     1081 cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc tgacccccaa
     1141 cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta
     1201 cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct
     1261 ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga
     1321 gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga
     1381 cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt
     1441 cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga
     1501 gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt
     1561 gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc
     1621 ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc
     1681 attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta
     1741 cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga
     1801 aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt
     1861 catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca
     1921 cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac
     1981 cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct
     2041 gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa
     2101 aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct
     2161 gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga
     2221 aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat
     2281 gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct
     2341 gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg
     2401 ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag
     2461 aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc
     2521 ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc
     2581 cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat
     2641 gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca
     2701 gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct
     2761 gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct
     2821 gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa
     2881 ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctgg ccgacgactc
     2941 catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc
     3001 ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct
     3061 gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact
     3121 ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt
     3181 ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg
     3241 ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca
     3301 gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc
     3361 cgtcgtggga accgccctga tcaaaaagta ccctgcgctg gaaagcgagt tcgtgtacgg
     3421 cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa
     3481 ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac
     3541 cctggccaac ggcgagatcc ggaaggcgcc tctgatcgag acaaacggcg aaaccgggga
     3601 gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca
     3661 agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct
     3721 gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta
     3781 cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa
     3841 gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag
     3901 aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa
     3961 aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa
     4021 gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa
     4081 atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga
     4141 taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga
     4201 gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct
     4261 gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca
     4321 cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat
     4381 cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag
     4441 catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acaaaaggcc
     4501 ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag gaattcggca gtggagaggg
     4561 cagaggaagt ctgctaacat gcggtgacgt cgaggagaat cctggcccaa tgaccgagta
     4621 caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc
     4681 cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga
     4741 gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt
     4801 gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc
     4861 gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc
     4921 cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt
     4981 cctggccacc gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt
     5041 gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc
     5101 gccccacaac ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt
     5161 gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgag aattctaact
     5221 agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     5281 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     5341 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     5401 caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga gcggccgcag
     5461 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     5521 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
     5581 gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     5641 atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg
     5701 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg
     5761 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     5821 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     5881 aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     5941 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     6001 tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt
     6061 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     6121 ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     6181 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     6241 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     6301 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     6361 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     6421 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     6481 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     6541 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     6601 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     6661 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     6721 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     6781 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     6841 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     6901 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     6961 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     7021 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     7081 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     7141 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     7201 ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc
     7261 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     7321 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     7381 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     7441 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     7501 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     7561 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     7621 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     7681 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     7741 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     7801 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     7861 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     7921 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     7981 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     8041 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     8101 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     8161 ggttcctggc cttttgctgg ccttttgctc acatgtgagg gcctatttcc catgattcct
     8221 tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta
     8281 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     8341 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     8401 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgggtc ttcgagaaga
     8461 cctgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
     8521 agtggcaccg agtcggtgct tttttgtttt agagctagaa atagcaagtt aaaataaggc
     8581 tagtccgttt ttagcgcgtg cgccaattct gcagacaaat ggctctagag gtacccgtta
     8641 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     8701 caatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     8761 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     8821 atgacggtaa atggcccgcc tggcattgtg cccagtacat gaccttatgg gactttccta
     8881 cttggcagta catctacgta ttagtcatcg ctattaccat ggtcgaggtg agccccacgt
     8941 tctgcttcac tctccccatc tcccccccct ccccaccccc aattttgtat ttatttattt
     9001 tttaattatt ttgtgcagcg atgggggcgg gggc
//
