LOCUS       sequence-148181-       10243 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one CRISPR/Cas9 vector with CAG promoter for expression in 
            human ESC/iPSC.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10243)
  AUTHORS   Stoehr A, Kennedy L, Yang Y, Patel S, Lin Y, Linask KL, Fergusson M,
            Zhu J, Gucek M, Zou J, Murphy E
  TITLE     The ribosomal prolyl-hydroxylase OGFOD1 decreases during cardiac 
            differentiation and modulates translation and splicing.
  JOURNAL   JCI Insight. 2019 May 21;5. pii: 128496. doi: 
            10.1172/jci.insight.128496.
  PUBMED    31112528
REFERENCE   2  (bases 1 to 10243)
  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: "JCI 
            Insight."; date: "2019-05-21"; volume: "5. pii: 128496. doi"; pages:
            " 10.1172/jci.insight.128496"
COMMENT     Sequence Label: pCAG-SpCas9-GFP-U6-gRNA
FEATURES             Location/Qualifiers
     source          1..10243
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        523..826
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        828..1105
                     /label=chicken beta-actin promoter
     intron          1107..2124
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2048..2070
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2132..2151
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2199..2208
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2208..2273
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2280..2300
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2325..6425
                     /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             2325..6425
                     /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             6426..6473
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6489..6542
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(6543..6545,6546..7256)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(6543..6545,6546..7256)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(6846..6865)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7193..7214
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(7284..7301)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7290..7497
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7506..7646
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7506..7635
                     /label=AAV2 ITR
     rep_origin      7721..8176
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7808..7827)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8018..8039
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8193..8212)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8312..8334
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8372..8390)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8458..8562
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8563..8631,8632..9423)
                     /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(8781..8800)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9594..10182
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10083..10102
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agtaacggcc gccagtgtgc tggaattcag gctagttatt aatagtaatc
      481 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      541 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      601 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      661 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      721 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      781 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc
      841 cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt
      901 tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggggc gcgcgccagg
      961 cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa
     1021 tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta
     1081 taaaaagcga agcgcgcggc gggcggggag tcgctgcgac gctgccttcg ccccgtgccc
     1141 cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag
     1201 gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg
     1261 cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc
     1321 ggggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc cgcgtgcggc
     1381 tccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc
     1441 agtgtgcgcg aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg gggctgcgag
     1501 gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg
     1561 tcggtcgggc tgcaaccccc cctgcacccc cctccccgag ttgctgagca cggcccggct
     1621 tcgggtgcgg ggctccgtac ggggcgtggc gcggggctcg ccgtgccggg cggggggtgg
     1681 cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg ccggggaggg ctcgggggag
     1741 gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg cggcgagccg cagccattgc
     1801 cttttatggt aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat ctgtgcggag
     1861 ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc gcggggcgaa gcggtgcggc
     1921 gccggcagga aggaaatggg cggggagggc cttcgtgcgt cgccgcgccg ccgtcccctt
     1981 ctccctctcc agcctcgggg ctgtccgcgg ggggacggct gccttcgggg gggacggggc
     2041 agggcggggt tcggcttctg gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt
     2101 catgccttct tctttttcct acagctcctg ggcaacgtgc tggttattgt gctgtctcat
     2161 cattttggca aagaattcct gaattctgca gatccggtgc caccatggac tataaggacc
     2221 acgacggaga ctacaaggat catgatattg attacaaaga cgatgacgat aagatggccc
     2281 caaagaagaa gcggaaggtc ggtatccacg gagtcccagc agccgacaag aagtacagca
     2341 tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg
     2401 tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc
     2461 tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg ctgaagagaa
     2521 ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca
     2581 gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg
     2641 tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg
     2701 cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg
     2761 acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc
     2821 acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc
     2881 agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg
     2941 acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa aatctgatcg
     3001 cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg
     3061 gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa ctgcagctga
     3121 gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg
     3181 ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga
     3241 gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag agatacgacg
     3301 agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt
     3361 acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt gacggcggag
     3421 ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg
     3481 aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg accttcgaca
     3541 acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg
     3601 aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc
     3661 gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca
     3721 gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac aagggcgctt
     3781 ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc aacgagaagg
     3841 tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag
     3901 tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag cagaaaaagg
     3961 ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg
     4021 actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt
     4081 tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc
     4141 tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg
     4201 aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag
     4261 tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc cggaagctga
     4321 tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg
     4381 gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc tttaaagagg
     4441 acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc
     4501 tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc
     4561 tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga
     4621 accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg
     4681 gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc
     4741 agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg
     4801 aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct cagagctttc
     4861 tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac cggggcaaga
     4921 gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg cggcagctgc
     4981 tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc gagagaggcg
     5041 gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa acccggcaga
     5101 tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaatg
     5161 acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc
     5221 ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg
     5281 cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg
     5341 agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc
     5401 aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg aactttttca
     5461 agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg
     5521 gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc
     5581 tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca
     5641 aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag aaggactggg
     5701 accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg
     5761 ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg ctggggatca
     5821 ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct
     5881 acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg ttcgagctgg
     5941 aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga aacgaactgg
     6001 ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg
     6061 gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag cactacctgg
     6121 acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc
     6181 tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga gagcaggccg
     6241 agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc ttcaagtact
     6301 ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg gacgccaccc
     6361 tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct cagctgggag
     6421 gcgacaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa aaggaattcg
     6481 gcagtggaga gggcagagga agtctgctaa catgcggtga cgtcgaggag aatcctggcc
     6541 cagtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     6601 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     6661 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     6721 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     6781 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     6841 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     6901 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     6961 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     7021 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     7081 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     7141 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     7201 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaaggaat
     7261 tctaactaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     7321 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     7381 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     7441 ggtggggcag gacagcaagg gggaggattg ggaagagaat agcaggcatg ctggggagcg
     7501 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     7561 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     7621 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     7681 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     7741 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     7801 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     7861 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     7921 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     7981 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     8041 acaacactca accctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     8101 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     8161 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     8221 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     8281 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     8341 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     8401 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     8461 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     8521 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     8581 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     8641 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     8701 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     8761 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     8821 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     8881 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     8941 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     9001 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     9061 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     9121 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     9181 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     9241 tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca
     9301 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     9361 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     9421 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     9481 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     9541 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     9601 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     9661 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     9721 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     9781 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     9841 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     9901 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     9961 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
    10021 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
    10081 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
    10141 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
    10201 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt
//
