LOCUS       sequence-254829-        9320 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing SpCas9 and sgAAVS1-2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9320)
  AUTHORS   Li S, Prasanna X, Salo VT, Vattulainen I, Ikonen E
  TITLE     An efficient auxin-inducible degron system with low basal 
            degradation in human cells.
  JOURNAL   Nat Methods. 2019 Aug 26. pii: 10.1038/s41592-019-0512-x. doi: 
            10.1038/s41592-019-0512-x.
  PUBMED    31451765
REFERENCE   2  (bases 1 to 9320)
  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: "Nat 
            Methods. 2019 Aug 26. pii: 10.1038/s41592-019-0512-x. doi: 
            10.1038/s41592-019-0512-x."
COMMENT     Sequence Label: pCas9-sgAAVS1-2
FEATURES             Location/Qualifiers
     source          1..9320
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(14..31)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(185..773)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(265..284)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(944..1735,1736..1804))
                     /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     1567..1586
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1805..1909)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1962..2210
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1962..1982
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2133..2152
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2231..2306
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        2464..2843
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2844..3047
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2998..3018
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3092..3111
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3092..3110
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      3140..3149
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3149..3214
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3221..3241
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3266..7366
                     /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             3266..7366
                     /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             7367..7414
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7430..7483
                     /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             7484..8188
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(7626..7644)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             8204..8260
                     /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"
     CDS             8267..8866
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(8267..8286)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     8763..8783
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(9060..9079)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    9104..9151
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
       61 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
      121 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
      181 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
      241 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
      301 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
      361 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
      421 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
      481 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
      541 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
      601 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
      661 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
      721 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
      781 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
      841 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
      901 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
      961 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     1021 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     1081 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     1141 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     1201 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     1261 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     1321 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     1381 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     1441 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     1501 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     1561 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     1621 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     1681 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     1741 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     1801 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     1861 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     1921 gaaaagtgcc acctgacggt accgctagcg tcgacggatc cgagggccta tttcccatga
     1981 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga
     2041 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt
     2101 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa
     2161 agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc gtcaccaatc
     2221 ctgtccctag gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac
     2281 ttgaaaaagt ggcaccgagt cggtgctttt tttagatctc gagtctagag gaggtcgctg
     2341 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga
     2401 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc
     2461 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
     2521 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     2581 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     2641 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     2701 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     2761 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     2821 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
     2881 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
     2941 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
     3001 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta
     3061 gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag
     3121 ctgcccggga tccaccggtg ccaccatgga ctataaggac cacgacggag actacaagga
     3181 tcatgatatt gattacaaag acgatgacga taagatggcc ccaaagaaga agcggaaggt
     3241 cggtatccac ggagtcccag cagccgacaa gaagtacagc atcggcctgg acatcggcac
     3301 caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa
     3361 ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag ccctgctgtt
     3421 cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac
     3481 cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga tggccaaggt
     3541 ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca
     3601 cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc
     3661 caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct
     3721 gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga
     3781 cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc agacctacaa
     3841 ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc
     3901 tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc ccggcgagaa
     3961 gaagaatggc ctgttcggaa acctgattgc cctgagcctg ggcctgaccc ccaacttcaa
     4021 gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca cctacgacga
     4081 cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt ttctggccgc
     4141 caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca ccgagatcac
     4201 caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc aggacctgac
     4261 cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga ttttcttcga
     4321 ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg aagagttcta
     4381 caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc tcgtgaagct
     4441 gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca tcccccacca
     4501 gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt acccattcct
     4561 gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct actacgtggg
     4621 ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg aggaaaccat
     4681 caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga gcttcatcga
     4741 gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca agcacagcct
     4801 gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg
     4861 aatgagaaag cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt
     4921 caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca agaaaatcga
     4981 gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac
     5041 ataccacgat ctgctgaaaa ttatcaagga caaggacttc ctggacaatg aggaaaacga
     5101 ggacattctg gaagatatcg tgctgaccct gacactgttt gaggacagag agatgatcga
     5161 ggaacggctg aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg
     5221 gcggagatac accggctggg gcaggctgag ccggaagctg atcaacggca tccgggacaa
     5281 gcagtccggc aagacaatcc tggatttcct gaagtccgac ggcttcgcca acagaaactt
     5341 catgcagctg atccacgacg acagcctgac ctttaaagag gacatccaga aagcccaggt
     5401 gtccggccag ggcgatagcc tgcacgagca cattgccaat ctggccggca gccccgccat
     5461 taagaagggc atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg
     5521 gcacaagccc gagaacatcg tgatcgaaat ggccagagag aaccagacca cccagaaggg
     5581 acagaagaac agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag agctgggcag
     5641 ccagatcctg aaagaacacc ccgtggaaaa cacccagctg cagaacgaga agctgtacct
     5701 gtactacctg cagaatgggc gggatatgta cgtggaccag gaactggaca tcaaccggct
     5761 gtccgactac gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg actccatcga
     5821 caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg tgccctccga
     5881 agaggtcgtg aagaagatga agaactactg gcggcagctg ctgaacgcca agctgattac
     5941 ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg aactggataa
     6001 ggccggcttc atcaagagac agctggtgga aacccggcag atcacaaagc acgtggcaca
     6061 gatcctggac tcccggatga acactaagta cgacgagaat gacaagctga tccgggaagt
     6121 gaaagtgatc accctgaagt ccaagctggt gtccgatttc cggaaggatt tccagtttta
     6181 caaagtgcgc gagatcaaca actaccacca cgcccacgac gcctacctga acgccgtcgt
     6241 gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt acggcgacta
     6301 caaggtgtac gacgtgcgga agatgatcgc caagagcgag caggaaatcg gcaaggctac
     6361 cgccaagtac ttcttctaca gcaacatcat gaactttttc aagaccgaga ttaccctggc
     6421 caacggcgag atccggaagc ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt
     6481 gtgggataag ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa
     6541 tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc aaagagtcta tcctgcccaa
     6601 gaggaacagc gataagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg
     6661 cttcgacagc cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa
     6721 gtccaagaaa ctgaagagtg tgaaagagct gctggggatc accatcatgg aaagaagcag
     6781 cttcgagaag aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga
     6841 cctgatcatc aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat
     6901 gctggcctct gccggcgaac tgcagaaggg aaacgaactg gccctgccct ccaaatatgt
     6961 gaacttcctg tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga
     7021 gcagaaacag ctgtttgtgg aacagcacaa gcactacctg gacgagatca tcgagcagat
     7081 cagcgagttc tccaagagag tgatcctggc cgacgctaat ctggacaaag tgctgtccgc
     7141 ctacaacaag caccgggata agcccatcag agagcaggcc gagaatatca tccacctgtt
     7201 taccctgacc aatctgggag cccctgccgc cttcaagtac tttgacacca ccatcgaccg
     7261 gaagaggtac accagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac
     7321 cggcctgtac gagacacgga tcgacctgtc tcagctggga ggcgacaaaa ggccggcggc
     7381 cacgaaaaag gccggccagg caaaaaagaa aaaggaattc ggcagtggag agggcagagg
     7441 aagtctgcta acatgcggtg acgtcgagga gaatcctggc ccagtgagca agggcgagga
     7501 ggataacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg agggctccgt
     7561 gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca
     7621 gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc
     7681 ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca tccccgacta
     7741 cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg
     7801 cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca tctacaaggt
     7861 gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga aaaccatggg
     7921 ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa
     7981 gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga ccacctacaa
     8041 ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt tggacatcac
     8101 ctcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg gccgccactc
     8161 caccggcggc atggacgagc tgtacaagga attcggaagc ggcgcaacaa acttctcact
     8221 actcaaacaa gcaggtgacg tggaggagaa tcccgggcct cttaagatga ccgagtacaa
     8281 gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc
     8341 cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg
     8401 ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg
     8461 ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg
     8521 ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc
     8581 gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct
     8641 ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct
     8701 ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc
     8761 ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc
     8821 cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgaatcg atgcgggact
     8881 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc
     8941 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     9001 atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg gaggctaact
     9061 gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag
     9121 aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg
     9181 cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt
     9241 tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg
     9301 gcaggccctg ccatagcctc
//
