LOCUS       sequence-426851-       10397 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express Cas9 with CAG promoter to improve the expression in hES 
            cells and sgRANGAP.C1 to tag endogenous RANGAP1 C-terminus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10397)
  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 10397)
  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-sgRANGAP.C1-CAG-Cas9-T2A-mCherry-P2A-Puro
FEATURES             Location/Qualifiers
     source          1..10397
                     /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..3122
                     /label=chicken beta-actin promoter
     intron          3123..4139
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     4063..4085
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4147..4166
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      4217..4226
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4226..4291
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4298..4318
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4343..8443
                     /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             4343..8443
                     /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             8444..8491
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             8507..8560
                     /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             8561..9265
                     /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(8703..8721)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             9281..9337
                     /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             9344..9943
                     /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(9344..9363)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     9840..9860
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(10137..10156)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    10181..10228
                     /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 gtctgctgca
     2221 gacgctgtac 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 ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc
     3001 ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt
     3061 ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg
     3121 cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc
     3181 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
     3241 ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg tggctgcgtg
     3301 aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg gggggtgcgt
     3361 gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc ggctgtgagc
     3421 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg agcgcggccg
     3481 ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa caaaggctgc gtgcggggtg
     3541 tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt cgggctgcaa ccccccctgc
     3601 acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtacggggc
     3661 gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg
     3721 cggggccgcc tcgggccggg gagggctcgg gggaggggcg cggcggcccc cggagcgccg
     3781 gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg tgcgagaggg
     3841 cgcagggact tcctttgtcc caaatctgtg cggagccgaa atctgggagg cgccgccgca
     3901 ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg
     3961 agggccttcg tgcgtcgccg cgccgccgtc cccttctccc tctccagcct cggggctgtc
     4021 cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg
     4081 tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc
     4141 tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa ttgctaccgg
     4201 actcagatcc accggtgcca ccatggacta taaggaccac gacggagact acaaggatca
     4261 tgatattgat tacaaagacg atgacgataa gatggcccca aagaagaagc ggaaggtcgg
     4321 tatccacgga gtcccagcag ccgacaagaa gtacagcatc ggcctggaca tcggcaccaa
     4381 ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt
     4441 gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggagccc tgctgttcga
     4501 cagcggcgaa acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag
     4561 acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga
     4621 cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga
     4681 gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac
     4741 catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat
     4801 ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct
     4861 gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca
     4921 gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc
     4981 cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa
     5041 gaatggcctg ttcggaaacc tgattgccct gagcctgggc ctgaccccca acttcaagag
     5101 caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct acgacgacga
     5161 cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa
     5221 gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa
     5281 ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg acctgaccct
     5341 gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca
     5401 gagcaagaac ggctacgccg gctacattga cggcggagcc agccaggaag agttctacaa
     5461 gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa
     5521 cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc cccaccagat
     5581 ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa
     5641 ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc
     5701 tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac
     5761 cccctggaac ttcgaggaag tggtggacaa gggcgcttcc gcccagagct tcatcgagcg
     5821 gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct
     5881 gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat
     5941 gagaaagccc gccttcctga gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa
     6001 gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg
     6061 cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata
     6121 ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga
     6181 cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga
     6241 acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg
     6301 gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc gggacaagca
     6361 gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca gaaacttcat
     6421 gcagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc
     6481 cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc ccgccattaa
     6541 gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga tgggccggca
     6601 caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc agaagggaca
     6661 gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca
     6721 gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc tgtacctgta
     6781 ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca accggctgtc
     6841 cgactacgat gtggaccata tcgtgcctca gagctttctg aaggacgact ccatcgacaa
     6901 caaggtgctg accagaagcg acaagaaccg gggcaagagc gacaacgtgc cctccgaaga
     6961 ggtcgtgaag aagatgaaga actactggcg gcagctgctg aacgccaagc tgattaccca
     7021 gagaaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac tggataaggc
     7081 cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg tggcacagat
     7141 cctggactcc cggatgaaca ctaagtacga cgagaatgac aagctgatcc gggaagtgaa
     7201 agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc agttttacaa
     7261 agtgcgcgag atcaacaact accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg
     7321 aaccgccctg atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg gcgactacaa
     7381 ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca aggctaccgc
     7441 caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta ccctggccaa
     7501 cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg
     7561 ggataagggc cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat
     7621 cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc tgcccaagag
     7681 gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt acggcggctt
     7741 cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa agggcaagtc
     7801 caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa gaagcagctt
     7861 cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga aaaaggacct
     7921 gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga agagaatgct
     7981 ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca aatatgtgaa
     8041 cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg ataatgagca
     8101 gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg agcagatcag
     8161 cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc tgtccgccta
     8221 caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc acctgtttac
     8281 cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca tcgaccggaa
     8341 gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga gcatcaccgg
     8401 cctgtacgag acacggatcg acctgtctca gctgggaggc gacaaaaggc cggcggccac
     8461 gaaaaaggcc ggccaggcaa aaaagaaaaa ggaattcggc agtggagagg gcagaggaag
     8521 tctgctaaca tgcggtgacg tcgaggagaa tcctggccca gtgagcaagg gcgaggagga
     8581 taacatggcc atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa
     8641 cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac
     8701 cgccaagctg aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc
     8761 tcagttcatg tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt
     8821 gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg
     8881 cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa
     8941 gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaaa ccatgggctg
     9001 ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca
     9061 gaggctgaag ctgaaggacg gcggccacta cgacgctgag gtcaagacca cctacaaggc
     9121 caagaagccc gtgcagctgc ccggcgccta caacgtcaac atcaagttgg acatcacctc
     9181 ccacaacgag gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac
     9241 cggcggcatg gacgagctgt acaaggaatt cggaagcggc gcaacaaact tctcactact
     9301 caaacaagca ggtgacgtgg aggagaatcc cgggcctctt aagatgaccg agtacaagcc
     9361 cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc
     9421 gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt
     9481 caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt
     9541 cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc
     9601 ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca
     9661 gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc
     9721 caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc
     9781 cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg
     9841 caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga
     9901 aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgaatcgatg cgggactctg
     9961 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
    10021 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
    10081 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa
    10141 acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat
    10201 aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac
    10261 tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc
    10321 ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca
    10381 ggccctgcca tagcctc
//
