LOCUS       sequence-273660-        9701 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty concatemer vector in which 2 sgRNAs can be inserted; with Cas9
            + GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9701)
  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 9701)
  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: "J Vis 
            Exp."; date: "2020-02-18"; volume: "(156). doi"; pages: " 
            10.3791/60704"
COMMENT     Sequence Label: px458_Conc2
FEATURES             Location/Qualifiers
     source          1..9701
                     /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"
     CDS             727..750
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        773..1021
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1040..1115
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             1121..1147
                     /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             1121..1147
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        1171..1419
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1440..1515
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        1579..1864
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        1866..2143
                     /label=chicken beta-actin promoter
     intron          2144..2371
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      2383..2392
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2392..2457
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2464..2484
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2509..6609
                     /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             2509..6609
                     /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             6610..6657
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6673..6726
                     /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(6727..6729,6730..7440)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(6727..6729,6730..7440)
                     /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(7030..7049)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7377..7398
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(7468..7485)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7474..7681
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7690..7830
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7690..7819
                     /label=AAV2 ITR
     rep_origin      7905..8360
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7992..8011)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8202..8223
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8377..8396)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8496..8518
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8556..8574)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8642..8746
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8747..8815,8816..9607)
                     /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(8965..8984)
                     /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 acatgtgact acaaggacga cgatgacaag gatctccaag gtcgggcagg aagagggcct
      781 atttcccatg attccttcat atttgcatat acgatacaag gctgttagag agataattag
      841 aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata
      901 atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac
      961 cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac
     1021 cggagtcttc gagaagacag ttttagagct agaaatagca agttaaaata aggctagtcc
     1081 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tacccctacg acgtgcccga
     1141 ctacgccgga tctccaaggt cgggcaggaa gagggcctat ttcccatgat tccttcatat
     1201 ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca
     1261 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt
     1321 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat
     1381 ttcttggctt tatatatctt gtggaaagga cgaaacaccg gaagtcttcg agaagacaag
     1441 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
     1501 gcaccgagtc ggtgcttttt tatggcatca atgcagaagc tgatctcaga ggaggacctg
     1561 ggatcctcta gaggtacccg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     1621 caacgacccc cgcccattga cgtcaatagt aacgccaata gggactttcc attgacgtca
     1681 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
     1741 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt gtgcccagta
     1801 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
     1861 catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc
     1921 cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg
     1981 gggggggcgc gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag
     2041 aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg
     2101 gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg
     2161 ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac
     2221 cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagct
     2281 gagcaagagg taagggttta agggatggtt ggttggtggg gtattaatgt ttaattacct
     2341 ggagcacctg cctgaaatca ctttttttca ggttggaccg gtgccaccat ggactataag
     2401 gaccacgacg gagactacaa ggatcatgat attgattaca aagacgatga cgataagatg
     2461 gccccaaaga agaagcggaa ggtcggtatc cacggagtcc cagcagccga caagaagtac
     2521 agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac
     2581 aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag
     2641 aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag
     2701 agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc
     2761 ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc
     2821 ctggtggaag aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag
     2881 gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc
     2941 accgacaagg ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg
     3001 ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc
     3061 atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc
     3121 gtggacgcca aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg
     3181 atcgcccagc tgcccggcga gaagaagaat ggcctgttcg gaaacctgat tgccctgagc
     3241 ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag
     3301 ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag
     3361 tacgccgacc tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc
     3421 ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac
     3481 gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag
     3541 aagtacaaag agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc
     3601 ggagccagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc
     3661 accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcggaccttc
     3721 gacaacggca gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg
     3781 caggaagatt tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc
     3841 ttccgcatcc cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg
     3901 accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc
     3961 gcttccgccc agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag
     4021 aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc
     4081 aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa
     4141 aaggccatcg tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa
     4201 gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat
     4261 cggttcaacg cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac
     4321 ttcctggaca atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg
     4381 tttgaggaca gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac
     4441 aaagtgatga agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag
     4501 ctgatcaacg gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc
     4561 gacggcttcg ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa
     4621 gaggacatcc agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc
     4681 aatctggccg gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac
     4741 gagctcgtga aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga
     4801 gagaaccaga ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa
     4861 gagggcatca aagagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag
     4921 ctgcagaacg agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac
     4981 caggaactgg acatcaaccg gctgtccgac tacgatgtgg accatatcgt gcctcagagc
     5041 tttctgaagg acgactccat cgacaacaag gtgctgacca gaagcgacaa gaaccggggc
     5101 aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag
     5161 ctgctgaacg ccaagctgat tacccagaga aagttcgaca atctgaccaa ggccgagaga
     5221 ggcggcctga gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg
     5281 cagatcacaa agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag
     5341 aatgacaagc tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat
     5401 ttccggaagg atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac
     5461 gacgcctacc tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa
     5521 agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc
     5581 gagcaggaaa tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt
     5641 ttcaagaccg agattaccct ggccaacggc gagatccgga agcggcctct gatcgagaca
     5701 aacggcgaaa ccggggagat cgtgtgggat aagggccggg attttgccac cgtgcggaaa
     5761 gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc
     5821 agcaaagagt ctatcctgcc caagaggaac agcgataagc tgatcgccag aaagaaggac
     5881 tgggacccta agaagtacgg cggcttcgac agccccaccg tggcctattc tgtgctggtg
     5941 gtggccaaag tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg
     6001 atcaccatca tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag
     6061 ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag
     6121 ctggaaaacg gccggaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgaa
     6181 ctggccctgc cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg
     6241 aagggctccc ccgaggataa tgagcagaaa cagctgtttg tggaacagca caagcactac
     6301 ctggacgaga tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct
     6361 aatctggaca aagtgctgtc cgcctacaac aagcaccggg ataagcccat cagagagcag
     6421 gccgagaata tcatccacct gtttaccctg accaatctgg gagcccctgc cgccttcaag
     6481 tactttgaca ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc
     6541 accctgatcc accagagcat caccggcctg tacgagacac ggatcgacct gtctcagctg
     6601 ggaggcgaca aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa gaaaaaggaa
     6661 ttcggcagtg gagagggcag aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct
     6721 ggcccagtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     6781 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     6841 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     6901 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     6961 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     7021 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     7081 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     7141 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     7201 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     7261 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     7321 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     7381 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     7441 gaattctaac tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     7501 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     7561 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     7621 gtggggtggg gcaggacagc aagggggagg attgggaaga gaatagcagg catgctgggg
     7681 agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc
     7741 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
     7801 tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc
     7861 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg
     7921 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     7981 cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     8041 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     8101 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
     8161 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     8221 tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat
     8281 ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     8341 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata
     8401 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     8461 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     8521 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     8581 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     8641 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     8701 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     8761 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     8821 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     8881 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     8941 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     9001 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     9061 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     9121 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     9181 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     9241 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     9301 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     9361 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     9421 tggctggttt attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc
     9481 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     9541 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     9601 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     9661 ttaatttaaa aggatctagg tgaagatcct ttttgataat c
//
