LOCUS       sequence-266294-       10104 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Empty concatemer vector in which 3 sgRNAs can be inserted; with Cas9
            + GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10104)
  AUTHORS   Gaebler AM, Hennig A, Buczolich K, Weitz J, Welsch T, Stange DE, 
            Pape K
  TITLE     Universal and Efficient Electroporation Protocol for Genetic 
            Engineering of Gastrointestinal Organoids.
  JOURNAL   J Vis Exp. 2020 Feb 18;(156). doi: 10.3791/60704.
  PUBMED    32150173
REFERENCE   2  (bases 1 to 10104)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Vis 
            Exp."; date: "2020-02-18"; volume: "(156). doi"; pages: " 
            10.3791/60704"
COMMENT     Sequence Label: px458_Conc3
FEATURES             Location/Qualifiers
     source          1..10104
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      77..665
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     566..585
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        783..1031
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1048..1123
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             1130..1153
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        1176..1424
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1443..1518
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             1524..1550
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             1524..1550
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        1574..1822
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1843..1918
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        1982..2267
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        2269..2546
                     /label=chicken beta-actin promoter
     intron          2547..2774
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      2786..2795
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2795..2860
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2867..2887
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2912..7012
                     /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             2912..7012
                     /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             7013..7060
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7076..7129
                     /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             join(7130..7132,7133..7843)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(7130..7132,7133..7843)
                     /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(7433..7452)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7780..7801
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(7871..7888)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7877..8084
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   8093..8233
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   8093..8222
                     /label=AAV2 ITR
     rep_origin      8308..8763
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8395..8414)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8605..8626
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8780..8799)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8899..8921
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8959..8977)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        9045..9149
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9150..9218,9219..10010)
                     /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(9368..9387)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
       61 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
      121 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
      181 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
      241 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
      301 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
      361 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
      421 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
      481 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      541 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      601 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      661 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
      721 acatgtccag ccctcactcc ttctctaggc gccggaatta gatctccaag gtcgggcagg
      781 aagagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
      841 agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
      901 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
      961 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
     1021 gacgaaacac cgggtcttcg agaagacgtt ttagagctag aaatagcaag ttaaaataag
     1081 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg actacaagga
     1141 cgacgatgac aaggatctcc aaggtcgggc aggaagaggg cctatttccc atgattcctt
     1201 catatttgca tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa
     1261 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
     1321 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
     1381 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggagtc ttcgagaaga
     1441 cagttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa
     1501 gtggcaccga gtcggtgctt ttttacccct acgacgtgcc cgactacgcc ggatctccaa
     1561 ggtcgggcag gaagagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     1621 ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa
     1681 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     1741 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     1801 cttgtggaaa ggacgaaaca ccggaagtct tcgagaagac aagttttaga gctagaaata
     1861 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
     1921 ttttatggca tcaatgcaga agctgatctc agaggaggac ctgggatcct ctagaggtac
     1981 ccgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     2041 tgacgtcaat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     2101 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     2161 acgtcaatga cggtaaatgg cccgcctggc attgtgccca gtacatgacc ttatgggact
     2221 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtgagcc
     2281 ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat
     2341 ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg cgcgcgccag
     2401 gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca
     2461 atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct
     2521 ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc
     2581 gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg
     2641 tgagcgggcg ggacggccct tctcctccgg gctgtaatta gctgagcaag aggtaagggt
     2701 ttaagggatg gttggttggt ggggtattaa tgtttaatta cctggagcac ctgcctgaaa
     2761 tcactttttt tcaggttgga ccggtgccac catggactat aaggaccacg acggagacta
     2821 caaggatcat gatattgatt acaaagacga tgacgataag atggccccaa agaagaagcg
     2881 gaaggtcggt atccacggag tcccagcagc cgacaagaag tacagcatcg gcctggacat
     2941 cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa
     3001 attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct
     3061 gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag
     3121 atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc
     3181 caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa
     3241 gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa
     3301 gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct
     3361 gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga
     3421 gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac
     3481 ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat
     3541 cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg
     3601 cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc tgacccccaa
     3661 cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta
     3721 cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct
     3781 ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga
     3841 gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga
     3901 cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt
     3961 cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga
     4021 gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt
     4081 gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc
     4141 ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc
     4201 attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta
     4261 cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga
     4321 aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt
     4381 catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca
     4441 cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac
     4501 cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct
     4561 gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa
     4621 aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct
     4681 gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga
     4741 aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat
     4801 gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct
     4861 gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg
     4921 ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag
     4981 aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc
     5041 ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc
     5101 cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat
     5161 gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca
     5221 gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct
     5281 gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct
     5341 gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa
     5401 ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga aggacgactc
     5461 catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc
     5521 ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct
     5581 gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact
     5641 ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt
     5701 ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg
     5761 ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca
     5821 gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc
     5881 cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg
     5941 cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa
     6001 ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac
     6061 cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaaccgggga
     6121 gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca
     6181 agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct
     6241 gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta
     6301 cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa
     6361 gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag
     6421 aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa
     6481 aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa
     6541 gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa
     6601 atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga
     6661 taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga
     6721 gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct
     6781 gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca
     6841 cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat
     6901 cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag
     6961 catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acaaaaggcc
     7021 ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag gaattcggca gtggagaggg
     7081 cagaggaagt ctgctaacat gcggtgacgt cgaggagaat cctggcccag tgagcaaggg
     7141 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     7201 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     7261 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     7321 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     7381 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     7441 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     7501 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     7561 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     7621 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     7681 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     7741 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     7801 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aaggaattct aactagagct
     7861 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     7921 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     7981 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     8041 agcaaggggg aggattggga agagaatagc aggcatgctg gggagcggcc gcaggaaccc
     8101 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     8161 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     8221 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     8281 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     8341 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt
     8401 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     8461 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     8521 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     8581 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact
     8641 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa
     8701 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     8761 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     8821 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     8881 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     8941 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     9001 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     9061 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     9121 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     9181 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     9241 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     9301 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     9361 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     9421 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     9481 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     9541 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     9601 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     9661 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     9721 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     9781 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     9841 ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg
     9901 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     9961 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
    10021 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
    10081 aggtgaagat cctttttgat aatc
//
