LOCUS       sequence-223638-        9787 bp    DNA     circular SYN 14-APR-2025
DEFINITION  CRISPR knockout, expresses Cas9, and sgRNA targeting AAVS1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9787)
  AUTHORS   Hojland Knudsen C, Asgrimsdottir ES, Rahimi K, Gill KP, Frandsen S, 
            Hvolbol Buchholdt S, Chen M, Kjems J, Febbraro F, Denham M
  TITLE     A Modified Monomeric Red Fluorescent Protein Reporter for Assessing 
            CRISPR Activity.
  JOURNAL   Front Cell Dev Biol. 2018 May 15;6:54. doi: 
            10.3389/fcell.2018.00054. eCollection 2018.
  PUBMED    29868584
REFERENCE   2  (bases 1 to 9787)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Front Cell 
            Dev Biol."; date: "2018-05-15"; volume: "6:54. doi"; pages: " 
            10.3389/fcell.2018.00054. eCollection 2018"
COMMENT     Sequence Label: hAAVS1-gs4_EF1a-Cas9-2A-GFP
FEATURES             Location/Qualifiers
     source          1..9787
                     /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        271..346
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        544..1722
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          775..1713
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1670..1690
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1743..1752
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1752..1817
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1824..1844
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1869..5969
                     /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             1869..5969
                     /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             5970..6017
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6033..6086
                     /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(6087..6089,6090..6800)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(6087..6089,6090..6800)
                     /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(6390..6409)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6737..6758
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6828..6845)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6834..7041
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7050..7190
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7050..7179
                     /label=AAV2 ITR
     rep_origin      7265..7720
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7352..7371)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7562..7583
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7737..7756)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7856..7878
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7916..7934)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8002..8106
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8107..8175,8176..8967)
                     /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(8325..8344)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9138..9726
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9627..9646
                     /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 tgggggttag acccaatatc gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga
      361 gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca
      421 gacaaatggc tctagaggta ccatcgatgt cgacgataag ctttgcaaag atggataaag
      481 ttttaaacag agaggaatct ttgcagctaa tggaccttct aggtcttgaa aggagtggga
      541 attggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg
      601 gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa
      661 agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt
      721 gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtaagt
      781 gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt atggcccttg cgtgccttga
      841 attacttcca cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg
      901 tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc
      961 tggcctgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct
     1021 gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc
     1081 tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg
     1141 gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg
     1201 cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg
     1261 cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca
     1321 ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg
     1381 aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt
     1441 ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc
     1501 gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg
     1561 atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg
     1621 taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag
     1681 acagtggttc aaagtttttt tcttccattt caggtgtcgt gaggaatttc gacatttaaa
     1741 tcgccaccat ggactataag gaccacgacg gagactacaa ggatcatgat attgattaca
     1801 aagacgatga cgataagatg gccccaaaga agaagcggaa ggtcggtatc cacggagtcc
     1861 cagcagccga caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg
     1921 ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg
     1981 accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag
     2041 ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga
     2101 tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc
     2161 acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct
     2221 tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga
     2281 gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg
     2341 cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca
     2401 gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa
     2461 accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga
     2521 gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg
     2581 gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg
     2641 ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc
     2701 tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg
     2761 ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg
     2821 cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg
     2881 tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct
     2941 acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca
     3001 tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc
     3061 tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc
     3121 tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa
     3181 agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa
     3241 acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg
     3301 aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg
     3361 ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca
     3421 ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct
     3481 tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag
     3541 tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg
     3601 aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga
     3661 aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata
     3721 tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct
     3781 atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct
     3841 ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa
     3901 tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg
     3961 acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata
     4021 gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc
     4081 agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca
     4141 tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg
     4201 agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac
     4261 accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg
     4321 ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg
     4381 accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca
     4441 gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga
     4501 tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca
     4561 atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga
     4621 gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga
     4681 tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga
     4741 agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca
     4801 acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca
     4861 aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc
     4921 ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct
     4981 acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga
     5041 agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg
     5101 attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg
     5161 aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc
     5221 tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg
     5281 tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga
     5341 gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag aagaatccca
     5401 tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc
     5461 ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg
     5521 aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg
     5581 ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg
     5641 tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga
     5701 gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg
     5761 ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg
     5821 gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca
     5881 ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac
     5941 ggatcgacct gtctcagctg ggaggcgaca aaaggccggc ggccacgaaa aaggccggcc
     6001 aggcaaaaaa gaaaaaggaa ttcggcagtg gagagggcag aggaagtctg ctaacatgcg
     6061 gtgacgtcga ggagaatcct ggcccagtga gcaagggcga ggagctgttc accggggtgg
     6121 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     6181 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     6241 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     6301 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     6361 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     6421 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     6481 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     6541 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     6601 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     6661 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     6721 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     6781 gcatggacga gctgtacaag gaattctaac tagagctcgc tgatcagcct cgactgtgcc
     6841 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     6901 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     6961 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     7021 gaatagcagg catgctgggg agcggccgca ggaaccccta gtgatggagt tggccactcc
     7081 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
     7141 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat
     7201 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca
     7261 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     7321 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     7381 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     7441 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
     7501 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     7561 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta ttcttttgat
     7621 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     7681 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca
     7741 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     7801 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     7861 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     7921 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     7981 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     8041 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     8101 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     8161 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     8221 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     8281 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     8341 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     8401 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     8461 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     8521 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     8581 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     8641 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     8701 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     8761 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     8821 ggaagccgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     8881 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     8941 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     9001 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     9061 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     9121 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     9181 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     9241 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     9301 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     9361 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     9421 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     9481 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     9541 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     9601 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     9661 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     9721 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     9781 cacatgt
//
