LOCUS       sequence-440854-        9647 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses Cas9-hGem and guideRNA for N terminal tagging of NIPBL.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9647)
  AUTHORS   Batty P, Langer CC, Takacs Z, Tang W, Blaukopf C, Peters JM, Gerlich
            DW
  TITLE     Cohesin-mediated DNA loop extrusion resolves sister chromatids in G2
            phase.
  JOURNAL   EMBO J. 2023 Aug 15;42(16):e113475. doi: 10.15252/embj.2023113475. 
            Epub 2023 Jun 26.
  PUBMED    37357575
REFERENCE   2  (bases 1 to 9647)
  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: "EMBO J."; 
            date: "2023-08-15"; volume: "42(16):e113475. doi"; pages: " 
            10.15252/embj.2023113475. Epub 2023 Jun 26"
COMMENT     Sequence Label: gRNA3_NIPBL_Cas9-hGem-for_N-terminal_tagging
FEATURES             Location/Qualifiers
     source          1..9647
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             42..98
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     regulatory      114..123
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             120..830
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(265..283)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(855..872)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    861..1068
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   1077..1217
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1077..1206
                     /label=AAV2 ITR
     rep_origin      1292..1747
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1379..1398)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1589..1610
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1764..1783)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1883..1905
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1943..1961)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2029..2133
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2134..2202,2203..2994)
                     /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(2352..2371)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3165..3753
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3654..3673
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3815..4063
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3815..3835
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3986..4005
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4085..4160
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        4257..4542
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          4824..5051
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      5063..5072
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5072..5137
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5144..5164
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5189..9289
                     /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             5189..9289
                     /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             9290..9337
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             join(9350..9647,1..32)
                     /codon_start=1
                     /product="degron consisting of residues 1-110 of human 
                     Geminin (Zielke and Edgar, 2015)"
                     /label=Gem1 (1-110)
                     /translation="MNPSMKQKQEEIKENIKNSSVPRRTLKMIQPSASGSLVGRENELS
                     AGLSKRKHRNDHLTSTTSSPGVIVPESSENKNLGGVTQESFDLMIKENPSSQYWKEVAE
                     KRRKAL"
ORIGIN
        1 aggaagtggc agaaaaacgg agaaaggcgc tgggaagcgg agctactaac ttcagcctgc
       61 tgaagcaggc tggagacgtg gaggagaacc ctggacctga attcatgccg gtcgccacca
      121 tggtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg cgcttcaagg
      181 tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc
      241 gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc cccctgccct
      301 tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac gtgaagcacc
      361 ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg
      421 tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg
      481 gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa
      541 tgcagaagaa aaccatgggc tgggaggcct cctccgagcg gatgtacccc gaggacggcg
      601 ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac tacgacgctg
      661 aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca
      721 acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgaac
      781 gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaagtag gaattcctag
      841 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
      901 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
      961 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     1021 ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagc ggccgcagga
     1081 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     1141 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
     1201 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     1261 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     1321 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct
     1381 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     1441 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     1501 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     1561 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     1621 aactctatct cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg
     1681 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     1741 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     1801 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     1861 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     1921 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     1981 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     2041 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     2101 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     2161 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     2221 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     2281 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     2341 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     2401 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     2461 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     2521 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     2581 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     2641 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     2701 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     2761 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     2821 gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca
     2881 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     2941 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     3001 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     3061 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     3121 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3181 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     3241 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     3301 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     3361 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     3421 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3481 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3541 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     3601 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     3661 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     3721 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     3781 ttcctggcct tttgctggcc ttttgctcac atgtgagggc ctatttccca tgattccttc
     3841 atatttgcat atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa
     3901 cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc
     3961 agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt
     4021 cgatttcttg gctttatata tcttgtggaa aggacgaaac accgtgtccc cattactact
     4081 cttggtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
     4141 aagtggcacc gagtcggtgc ttttttgttt tagagctaga aatagcaagt taaaataagg
     4201 ctagtccgtt tttagcgcgt gcgccaattc tgcagacaaa tggctctaga ggtacccgtt
     4261 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
     4321 tcaatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     4381 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     4441 aatgacggta aatggcccgc ctggcattgt gcccagtaca tgaccttatg ggactttcct
     4501 acttggcagt acatctacgt attagtcatc gctattacca tggtcgaggt gagccccacg
     4561 ttctgcttca ctctccccat ctcccccccc tccccacccc caattttgta tttatttatt
     4621 ttttaattat tttgtgcagc gatgggggcg gggggggggg gggggcgcgc gccaggcggg
     4681 gcgggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggccccagg
     4741 gggggggggc ccccgagagt tctctttttt gggcgggggg gggggggggg gggccctaaa
     4801 aaaagagaag gagggggggg gggggagtcg ctgcgcgctg ccttcgcccc gtgccccgct
     4861 ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga
     4921 gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg taagggttta
     4981 agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg cctgaaatca
     5041 ctttttttca ggttggaccg gtgccaccat ggactataag gaccacgacg gagactacaa
     5101 ggatcatgat attgattaca aagacgatga cgataagatg gccccaaaga agaagcggaa
     5161 ggtcggtatc cacggagtcc cagcagccga caagaagtac agcatcggcc tggacatcgg
     5221 caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt
     5281 caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct
     5341 gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata
     5401 caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa
     5461 ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa
     5521 gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta
     5581 ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg
     5641 gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg
     5701 cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta
     5761 caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct
     5821 gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga
     5881 gaagaagaat ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt
     5941 caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga
     6001 cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc
     6061 cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat
     6121 caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct
     6181 gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt
     6241 cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt
     6301 ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa
     6361 gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca
     6421 ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt
     6481 cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt
     6541 gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac
     6601 catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat
     6661 cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag
     6721 cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga
     6781 gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct
     6841 gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat
     6901 cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg
     6961 cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa
     7021 cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat
     7081 cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa
     7141 gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga
     7201 caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa
     7261 cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca
     7321 ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc
     7381 cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg
     7441 ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa
     7501 gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg
     7561 cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta
     7621 cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg
     7681 gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat
     7741 cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc
     7801 cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat
     7861 tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga
     7921 taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc
     7981 acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga
     8041 agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt
     8101 ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt
     8161 cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga
     8221 ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc
     8281 taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct
     8341 ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat
     8401 cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt
     8461 gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc
     8521 caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg
     8581 cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg
     8641 caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag
     8701 cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa
     8761 ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag
     8821 aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata
     8881 tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa
     8941 tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca
     9001 gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc
     9061 cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct
     9121 gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga
     9181 ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat
     9241 caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca aaaggccggc
     9301 ggccacgaaa aaggccggcc aggcaaaaaa gaaaaaggga tccaagctta tgaatcccag
     9361 tatgaagcag aaacaagaag aaatcaaaga gaatataaag aatagttctg tcccaagaag
     9421 aactctgaag atgattcagc cttctgcatc tggatctctt gttggaagag aaaatgagct
     9481 gtccgcaggc ttgtccaaaa ggaaacatcg gaatgaccac ttaacatcta caacttccag
     9541 ccctggggtt attgtcccag aatctagtga aaataaaaat cttggaggag tcacccagga
     9601 gtcatttgat cttatgatta aagaaaatcc atcctctcag tattgga
//
