LOCUS       sequence-318845-       10244 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To generate HSD17B11 KO in human cells. Co-expresses eSpCas9(1.1), 
            GFP and a guide RNA against HSD17B11 exon 1..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10244)
  AUTHORS   Demange P, Joly E, Marcoux J, Zanon PRA, Listunov D, Rulliere P, 
            Barthes C, Noirot C, Izquierdo JB, Rozie A, Pradines K, Hee R, de 
            Brito MV, Marcellin M, Serre RF, Bouchez O, Burlet-Schiltz O, 
            Oliveira MCF, Ballereau S, Bernardes-Genisson V, Maraval V, Calsou 
            P, Hacker SM, Genisson Y, Chauvin R, Britton S
  TITLE     SDR enzymes oxidize specific lipidic alkynylcarbinols into cytotoxic
            protein-reactive species.
  JOURNAL   Elife. 2022 May 10;11. pii: 73913. doi: 10.7554/eLife.73913.
  PUBMED    35535493
REFERENCE   2  (bases 1 to 10244)
  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: "Elife."; 
            date: "2022-05-10"; volume: "11. pii: 73913. doi"; pages: " 
            10.7554/eLife.73913"
COMMENT     Sequence Label: pCAG-eSpCas9-2A-GFP-sgRNA-HSD17B11
FEATURES             Location/Qualifiers
     source          1..10244
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     315..334
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        476..724
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     476..496
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     647..666
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        746..821
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        1001..1304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          1584..2600
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2524..2546
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2608..2627
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2675..2684
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2684..2749
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2756..2776
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2801..5338,5339..5341,5342..5803,5804..5806,5807..5974,
                     5975..5977,5978..6901)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Slaymaker et al., 2016)"
                     /label=eSpCas9(1.1)
                     /note="carries the mutations K848A, K1003A, and R1060A"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6902..6949
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6965..7018
                     /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(7019..7021,7022..7732)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(7019..7021,7022..7732)
                     /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(7322..7341)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7669..7690
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(7760..7777)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7766..7973
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7982..8122
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7982..8111
                     /label=AAV2 ITR
     rep_origin      8197..8652
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8284..8303)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8494..8515
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8669..8688)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8788..8810
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8848..8866)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8934..9038
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9039..9107,9108..9899)
                     /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(9257..9276)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(10070..10244,1..414)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
       61 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
      121 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
      181 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
      241 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
      301 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
      361 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
      421 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgtgaggg
      481 cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta gagagataat
      541 tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta
      601 ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct
      661 taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa
      721 caccgtgtaa tcagcacgat ttcgcgtttt agagctagaa atagcaagtt aaaataaggc
      781 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttgtt ttagagctag
      841 aaatagcaag ttaaaataag gctagtccgt ttttagcgcg tgcgccaatt ctgcagacaa
      901 atggctctag taacggccgc cagtgtgctg gaattcaggc tagttattaa tagtaatcaa
      961 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
     1021 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     1081 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     1141 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     1201 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     1261 ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca
     1321 cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg tatttattta
     1381 ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggsgs gcgcsrggsg
     1441 gggsggggsg gggsgrgggg sggggsgggg sgrggsggrg rggkgsggcg gcagccaatc
     1501 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata
     1561 aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct
     1621 ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga
     1681 gcgggcggga cggcccttct cctccgggct gtaattagcg cttggtttaa tgacggcttg
     1741 tttcttttct gtggctgcgt gaaagccttg aggggctccg ggagggccct ttgtgcgggg
     1801 ggagcggctc ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg
     1861 cgctgcccgg cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg
     1921 tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg cggggggggc tgcgagggga
     1981 acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg
     2041 tcgggctgca accccccctg cacccccctc cccgagttgc tgagcacggc ccggcttcgg
     2101 gtgcggggct ccgtacgggg cgtggcgcgg ggctcgccgt gccgggcggg gggkggcggc
     2161 aggkgggggt gccgggcggg gcggggccgc ckcgggccgg ggrgggctcg ggggrggggc
     2221 gcggcggccc ccggagcgcc ggcggctgtc gaggcgcggc gagccgcagc cattgccttt
     2281 tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc ccaaatctgt gcggagccga
     2341 aatctgggag gcgccgccgc accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg
     2401 gcaggaagga aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc
     2461 ctctccagcc tcggggctgt ccgcgggggg acggctgcct tcggggggga cggggcaggg
     2521 cggggttcgg cttctggcgt gtgaccggcg gctctagagc ctctgctaac catgttcatg
     2581 ccttcttctt tttcctacag ctcctgggca acgtgctggt tattgtgctg tctcatcatt
     2641 ttggcaaaga attcctgaat tctgcagatc cggtgccacc atggactata aggaccacga
     2701 cggagactac aaggatcatg atattgatta caaagacgat gacgataaga tggccccaaa
     2761 gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt acagcatcgg
     2821 cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc
     2881 cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat
     2941 cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc
     3001 cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa
     3061 cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga
     3121 agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta
     3181 ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa
     3241 ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt
     3301 cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct
     3361 ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc
     3421 caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca
     3481 gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct
     3541 gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa
     3601 ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga
     3661 cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt
     3721 gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca
     3781 ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa
     3841 agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag
     3901 ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga
     3961 actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg
     4021 cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga
     4081 tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat
     4141 cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa
     4201 gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc
     4261 ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct
     4321 gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa
     4381 atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat
     4441 cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta
     4501 cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa
     4561 cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga
     4621 caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga
     4681 cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat
     4741 gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa
     4801 cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt
     4861 cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat
     4921 ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc
     4981 cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt
     5041 gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca
     5101 gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat
     5161 caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa
     5221 cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact
     5281 ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctggc
     5341 cgacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga
     5401 caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa
     5461 cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct
     5521 gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac
     5581 aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa
     5641 gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa
     5701 ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta
     5761 cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctgcgctgg aaagcgagtt
     5821 cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga
     5881 aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac
     5941 cgagattacc ctggccaacg gcgagatccg gaaggcgcct ctgatcgaga caaacggcga
     6001 aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag
     6061 catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga
     6121 gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc
     6181 taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa
     6241 agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat
     6301 catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa
     6361 agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa
     6421 cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct
     6481 gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc
     6541 ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga
     6601 gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga
     6661 caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa
     6721 tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga
     6781 caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat
     6841 ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga
     6901 caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagg aattcggcag
     6961 tggagagggc agaggaagtc tgctaacatg cggtgacgtc gaggagaatc ctggcccagt
     7021 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     7081 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     7141 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     7201 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     7261 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     7321 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     7381 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     7441 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     7501 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     7561 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     7621 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     7681 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca aggaattcta
     7741 actagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     7801 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     7861 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     7921 gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg ggagcggccg
     7981 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     8041 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     8101 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     8161 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     8221 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc
     8281 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     8341 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     8401 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     8461 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     8521 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
     8581 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     8641 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     8701 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     8761 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     8821 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     8881 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     8941 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     9001 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     9061 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     9121 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     9181 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     9241 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     9301 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     9361 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     9421 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     9481 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     9541 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     9601 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     9661 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     9721 ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg
     9781 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     9841 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     9901 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     9961 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
    10021 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
    10081 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
    10141 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
    10201 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccac
//
