LOCUS       sequence-426855-       10398 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express Cas9 with CAG promoter to improve the expression in hES 
            cells and sgSEC61B.N1 to tag endogenous SEC61B N-terminus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10398)
  AUTHORS   Li S, Wang Y, van der Stoel M, Zhou X, Madhusudan S, Kanerva K, 
            Nguyen VD, Eskici N, Olkkonen VM, Zhou Y, Raivio T, Ikonen E
  TITLE     HiHo-AID2: boosting homozygous knock-in efficiency enables robust 
            generation of human auxin-inducible degron cells.
  JOURNAL   Genome Biol. 2024 Feb 26;25(1):58. doi: 10.1186/s13059-024-03187-w.
  PUBMED    38409044
REFERENCE   2  (bases 1 to 10398)
  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: "Genome 
            Biol."; date: "2024-02-26"; volume: "25(1):58. doi"; pages: " 
            10.1186/s13059-024-03187-w"
COMMENT     Sequence Label: pGL3-sgSEC61B.N1-CAG-Cas9-T2A-mCherry-P2A-Puro
FEATURES             Location/Qualifiers
     source          1..10398
                     /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        2232..2307
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        2465..2844
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2846..3123
                     /label=chicken beta-actin promoter
     intron          3124..4140
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     4064..4086
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4148..4167
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      4218..4227
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4227..4292
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4299..4319
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4344..8444
                     /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             4344..8444
                     /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             8445..8492
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             8508..8561
                     /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             8562..9266
                     /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(8704..8722)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             9282..9338
                     /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             9345..9944
                     /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(9345..9364)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     9841..9861
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(10138..10157)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    10182..10229
                     /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 gtgccaccct
     2221 catctccaat agttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
     2281 cttgaaaaag tggcaccgag tcggtgcttt ttttagatct cgagtctaga ggaggtcgct
     2341 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
     2401 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
     2461 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
     2521 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
     2581 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     2641 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     2701 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     2761 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     2821 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
     2881 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
     2941 gcgatggggg cggggggggg gggggggcgc gcgccaggcg gggcggggcg gggcgagggg
     3001 cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag
     3061 tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg
     3121 gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc
     3181 ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct
     3241 cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct gtggctgcgt
     3301 gaaagccttg aggggctccg ggagggccct ttgtgcgggg ggagcggctc ggggggtgcg
     3361 tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag
     3421 cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg gagcgcggcc
     3481 gggggcggtg ccccgcggtg cggggggggc tgcgagggga acaaaggctg cgtgcggggt
     3541 gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg tcgggctgca accccccctg
     3601 cacccccctc cccgagttgc tgagcacggc ccggcttcgg gtgcggggct ccgtacgggg
     3661 cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc aggtgggggt gccgggcggg
     3721 gcggggccgc ctcgggccgg ggagggctcg ggggaggggc gcggcggccc ccggagcgcc
     3781 ggcggctgtc gaggcgcggc gagccgcagc cattgccttt tatggtaatc gtgcgagagg
     3841 gcgcagggac ttcctttgtc ccaaatctgt gcggagccga aatctgggag gcgccgccgc
     3901 accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg
     3961 gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc tcggggctgt
     4021 ccgcgggggg acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt
     4081 gtgaccggcg gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag
     4141 ctcctgggca acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attgctaccg
     4201 gactcagatc caccggtgcc accatggact ataaggacca cgacggagac tacaaggatc
     4261 atgatattga ttacaaagac gatgacgata agatggcccc aaagaagaag cggaaggtcg
     4321 gtatccacgg agtcccagca gccgacaaga agtacagcat cggcctggac atcggcacca
     4381 actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag aaattcaagg
     4441 tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc ctgctgttcg
     4501 acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga agatacacca
     4561 gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg gccaaggtgg
     4621 acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat aagaagcacg
     4681 agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccca
     4741 ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac ctgcggctga
     4801 tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc gagggcgacc
     4861 tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag acctacaacc
     4921 agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg
     4981 ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc ggcgagaaga
     5041 agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc aacttcaaga
     5101 gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc tacgacgacg
     5161 acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt ctggccgcca
     5221 agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc gagatcacca
     5281 aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag gacctgaccc
     5341 tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt ttcttcgacc
     5401 agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa gagttctaca
     5461 agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc gtgaagctga
     5521 acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc ccccaccaga
     5581 tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac ccattcctga
     5641 aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac tacgtgggcc
     5701 ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag gaaaccatca
     5761 ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc ttcatcgagc
     5821 ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag cacagcctgc
     5881 tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg accgagggaa
     5941 tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac ctgctgttca
     6001 agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt
     6061 gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc ctgggcacat
     6121 accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag gaaaacgagg
     6181 acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag atgatcgagg
     6241 aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag ctgaagcggc
     6301 ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc cgggacaagc
     6361 agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac agaaacttca
     6421 tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa gcccaggtgt
     6481 ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc cccgccatta
     6541 agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc
     6601 acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc cagaagggac
     6661 agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag ctgggcagcc
     6721 agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag ctgtacctgt
     6781 actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc aaccggctgt
     6841 ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac tccatcgaca
     6901 acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg ccctccgaag
     6961 aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag ctgattaccc
     7021 agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg
     7081 ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac gtggcacaga
     7141 tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc cgggaagtga
     7201 aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca
     7261 aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtcgtgg
     7321 gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac ggcgactaca
     7381 aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg
     7441 ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt accctggcca
     7501 acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt
     7561 gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaata
     7621 tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc ctgcccaaga
     7681 ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct
     7741 tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt
     7801 ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct
     7861 tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc
     7921 tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc
     7981 tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc aaatatgtga
     8041 acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc
     8101 agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca
     8161 gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct
     8221 acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta
     8281 ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc atcgaccgga
     8341 agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag agcatcaccg
     8401 gcctgtacga gacacggatc gacctgtctc agctgggagg cgacaaaagg ccggcggcca
     8461 cgaaaaaggc cggccaggca aaaaagaaaa aggaattcgg cagtggagag ggcagaggaa
     8521 gtctgctaac atgcggtgac gtcgaggaga atcctggccc agtgagcaag ggcgaggagg
     8581 ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga
     8641 acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga
     8701 ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc
     8761 ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact
     8821 tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg
     8881 gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga
     8941 agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaaa accatgggct
     9001 gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc
     9061 agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg
     9121 ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct
     9181 cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca
     9241 ccggcggcat ggacgagctg tacaaggaat tcggaagcgg cgcaacaaac ttctcactac
     9301 tcaaacaagc aggtgacgtg gaggagaatc ccgggcctct taagatgacc gagtacaagc
     9361 ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg
     9421 cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg
     9481 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg
     9541 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg
     9601 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc
     9661 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg
     9721 ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc
     9781 ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc
     9841 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg
     9901 aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgaatcgat gcgggactct
     9961 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
    10021 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
    10081 cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga
    10141 aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa
    10201 taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca
    10261 ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc
    10321 cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc
    10381 aggccctgcc atagcctc
//
