LOCUS Exported 7476 bp ds-DNA circular SYN 01-MAY-2020 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7476) AUTHORS Alex Brown TITLE Direct Submission JOURNAL Exported Monday, May 11, 2020 from SnapGene Viewer 5.1.0 https://www.snapgene.com FEATURES Location/Qualifiers source 1..7476 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 104..208 /gene="bla" /label=AmpR promoter primer_bind 135..158 /label=MK 4294 /note="[-] pUC origin, Amp gene::pUC/Amp-ClaI. Together with 4293, this primer will amplify the pUC-Amp cassette, which will then be used to construct the DTA-based mIL-15 KIN construct." primer_bind 137..161 /label=MK 4644 /note="[-] Kanamycin::Kanamycin (HindIII)-3'. Togetherwith 4643 will amplify the Kanamycin gene which will have an Xba I at the 5' end and HindIII at the 3' end." promoter 139..167 /label=AmpR_promoter /note="/translation=MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLG ARVGY IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA EIGASLIKHW*" gene 209..1069 /gene="Ampicillin" /label=Ampicillin /note="/translation=MPLRAFNPVSSFRWARGMTIVAALMTVFFIMQLVGQVPA ALWVI FGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAIIAGMAADALGYVL LAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSLAALTSLTSITG PLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST*" CDS 209..1069 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(304..333) /label=MK 4680 /note="[-] DTA (Diphteria toxin-A)::DTA ORF#2-3'. Togetherwith 4665 will amplify the DTA gene cassette from the IL-15 Knock-IN construct. This PCR is used to test if the cassette makes functional gene for negative selection ==> none of the selected ES clones should have this cassette intact!" primer_bind complement(348..371) /label=MK 4666 /note="[-] DTA (Diphteria toxin-A)::DTA ORF#1-3'. Togetherwith 4665 will amplify the DTA gene cassette from the IL-15 Knock-IN construct. This PCR is used to test if the cassette makes functional gene for negative selection ==> none of the selected ES clones should have this cassette intact!" primer_bind complement(1049..1070) /label=MK 4889 /note="[+] AMP NEO BAC::PBAC108L(#1)-Amp-5'. Together with 4890 makes a 2.8 kb fragment containing Amp and NEO cassettes. Anneals to the Amp site. Use L2neo-IRES-EGFPpA plasmid from Nigel Killeen as a template. Use the PCR fragment for the next PCR reaction with primers 4891 x 4892 ==> this will add some more homology for BAC108L." rep_origin 1240..1828 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1884..1905 /label=MK 1727 /note="[+] pTz18R::From Afl3 site. Used to make pTMet" primer_bind 1907..1927 /label=MK 4553 /note="[] ::" primer_bind complement(1923..1957) /label=MK 4293 /note="[+] pUC origin, Amp gene::pUC/Amp-AvrII. Together with 4294, this primer will amplify the pUC-Amp cassette, which will then be used to construct the DTA-based mIL-15 KIN construct." misc_feature 2014..2156 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(2080..2099) /label=MK 1597 /note="[+] URA3::MNN9 k.o. primer" primer_bind complement(2080..2099) /label=MK 1595 /note="[+] URA3::PMR1 K.O. PRIMER" primer_bind complement(2080..2099) /label=MK 1510 /note="[+] URA3::KEX2 homology for gene disruption" misc_feature 2242..2264 /label=pGEX_3_primer /note="/translation=MPLRAFNPVSSFRWARGMTIVAALMTVFFIMQLVGQVPA ALWVI FGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAIIAGMAADALGYVL LAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSLAALTSLTSITG PLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST*" primer_bind 2242..2264 /label=MK 2420 /note="[-] reverse sequence primer for pGEX vector::found on pharmacia web site" misc_feature 2258..2449 /label=ROP /note="/translation=MPLRAFNPVSSFRWARGMTIVAALMTVFFIMQLVGQVPA ALWVI FGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAIIAGMAADALGYVL LAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSLAALTSLTSITG PLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST*" gene complement(3088..3702) /gene="tet (576 - 1190)" /label=tet (576 - 1190) /note="/translation=MPLRAFNPVSSFRWARGMTIVAALMTVFFIMQLVGQVPA ALWVI FGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAIIAGMAADALGYVL LAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSLAALTSLTSITG PLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST*" misc_binding 3397..3403 /label=MEME Motif 7 [+] /bound_moiety="MEME motif search from last 110bp of murine V-alpha V-beta transcripts " misc_binding 3548..3553 /label=MEME Motif 6 [+] /bound_moiety="MEME motif search from last 110bp of murine V-alpha V-beta transcripts " primer_bind 3643..3662 /label=MK 3348 /note="[-] pGL3 (luciferase vectors)::EC66; RVprimer4 for sequencing pGL3-Basic, 2080-2061, into SV40 enhancer; Works" primer_bind 3679..3698 /label=MK 4723 /note="[+] pBACe3.6::pBACe3.6 - sequencing-5'. Sequencing of the BAC vector." primer_bind complement(3721..3744) /label=MK 3805 /note="[] Litmus plasmid::Litmus Sequencing. Primer for sequencing Litmus plasmid" primer_bind complement(3721..3741) /label=MK 4798 /note="[-] GalK::pGalK-seq.#2-3. For sequencing of the pGalK plasmid in the reverse direction." CDS complement(3761..4843) /codon_start=1 /gene="lacI" /product="lac repressor" /label=lacI /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" primer_bind complement(3808..3824) /label=MK 1197 /note="[-] OS-A:NotI::OSdup-del Sequencing Primer-at Approx. 25" primer_bind 4805..4824 /label=MK 6810 /note="[-] LacI::LacI-R" promoter complement(4844..4921) /gene="lacI" /label=lacI promoter misc_feature 5127..5146 /label=pBRrevBam_primer primer_bind 5127..5146 /label=MK 6785 /note="[+] pET19b ORF::pBRrevBam" primer_bind 5229..5249 /label=MK 820 /note="[+] pTZ 234-255::reverse primer upto EcoRI site" promoter 5230..5248 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind 5231..5247 /label=MK 5415 /note="[] ::" primer_bind 5231..5247 /label=MK 2583 /note="[] T7 seq primer::T7" primer_bind 5242..5270 /label=MK 5558 /note="[+] newly made sequence primer 4405::" primer_bind 5242..5270 /label=MK 4405 /note="[+] pET30-BirA::5' Sequencing Oligo" primer_bind 5246..5265 /label=MK 4291 /note="[+] pET-30b vector::sequencing primer - 5' of mult cloning site of pET-30b (in the lac promoter region)" protein_bind 5249..5273 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 5249..5265 /label=MK 1726 /note="[+] pTz18R ::5' sequencing oligo for pTZ/pMet" primer_bind 5249..5263 /label=MK 1377 /note="[+] pK233::Sequencing/PCR oligo" primer_bind 5261..5281 /label=MK 1729 /note="[+] pET11d::Sequencing oligo" primer_bind complement(5281..5314) /label=MK 4401 /note="[-] pET30-BirA::Change MCS (2 of 2) PO4" misc_feature 5293..5309 /label=T7_transl_en_RBS primer_bind 5315..5333 /label=MK 6633 /note="[+] pET11d::Extended 6HIS 5' extension to insert (NcoI)" CDS 5329..5346 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 5356..5373 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS 5377..7152 /codon_start=1 /label=Bst Large Fragment ORF /translation="SHMTDEGEKPLAGMDFAIADSVTDEMLADKAALVVEVVGDNYHHA PIVGIALANERGRFFLRPETALADPKFLAWLGDETKKKTMFDSKRAAVALKWKGIELRG VVFDLLLAAYLLDPAQAAGDVAAVAKMHQYEAVRSDEAVYGKGAKRTVPDEPTLAEHLA RKAAAIWALEEPLMDELRRNEQDRLLTELEQPLAGILANMEFTGVKVDTKRLEQMGAEL TEQLQAVERRIYELAGQEFNINSPKQLGTVLFDKLQLPVLKKTKTGYSTSADVLEKLAP HHEIVEHILHYRQLGKLQSTYIEGLLKVVHPVTGKVHTMFNQALTQTGRLSSVEPNLQN IPIRLEEGRKIRQAFVPSEPDWLIFAADYSQIELRVLAHIAEDDNLIEAFRRGLDIHTK TAMDIFHVSEEDVTANMRRQAKAVNFGIVYGISDYGLAQNLNITRKEAAEFIERYFASF PGVKQYMDNIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERTAMNTPIQGSAAD IIKKAMIDLSVRLREERLQARLLLQVHDELILEAPKEEIERLCRLVPEVMEQAVALRVP LKVDYHYGPTWYDAK" primer_bind 5378..5405 /label=MK 6950 /note="[+] Bst Large Fragment::Addition of NdeI for cloning into pET 15b" CDS 5378..5383 /codon_start=1 /label=Bst Large Fragment ORF /translation="H" primer_bind complement(5947..5961) /label=MK 1528 /note="[-] IE beta mutant::Introduce 64Q>R" primer_bind complement(7130..7158) /label=MK 6951 /note="[-] Bst Large Fragment::Addition of BamHI for cloning into pET 15b" terminator 7161..7289 /label=T7_terminator primer_bind complement(7179..7199) /label=MK 1730 /note="[-] pET11d::Sequencing oligo" primer_bind complement(7181..7205) /label=MK 5625 /note="[-] pET30::3' sequencing Oligo" primer_bind complement(7202..7227) /label=MK 4735 /note="[-] tdTomato::pRSET vector sequencing. To sequence the vector in the reverse orientation." misc_feature 7203..7221 /label=T7_Terminal_primer primer_bind complement(7203..7221) /label=MK 6786 /note="[-] pET19b ORF::T7 Terminal Primer" primer_bind complement(7203..7221) /label=MK 4114 /note="[-] HELIOS (mouse)::T7 Rv Seq primer" terminator 7217..7264 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" primer_bind complement(7247..7274) /label=MK 6635 /note="[-] pET11d::3' sequencing oligo downstream of BlpI site" primer_bind 7404..7463 /label=MK 4891 /note="[+] AMP NEO BAC::PBAC108L(#2)--5'. Together with 4892 makes a 2.8 kb fragment, containing Amp and NEO cassettes, which can be used to target pBAC108L backbone through the recombineering. Use the PCR product obtained using 4889 x 4890 (see above) as a template. Will add some more homology for BAC108L." promoter complement(7439..7467) /label=tet promoter /note="E. coli promoter for tetracycline efflux protein gene" ORIGIN 1 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 61 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 121 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 181 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 241 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 301 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 361 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 421 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg 481 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 541 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 601 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 661 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 721 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact 781 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 841 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 901 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 961 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 1021 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 1081 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 1141 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 1201 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 1261 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 1321 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 1381 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 1441 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 1501 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 1561 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 1621 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 1681 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 1741 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 1801 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 1861 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 1921 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 1981 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca 2041 tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg ctctgatgcc 2101 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc 2161 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 2221 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 2281 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga 2341 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc 2401 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg 2461 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca 2521 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac 2581 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg 2641 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga 2701 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga 2761 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc 2821 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg 2881 tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccgaga 2941 tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg 3001 tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc 3061 cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg 3121 acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg ccaacccgtt 3181 ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccag tgatcgaagt 3241 taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt catctacctg 3301 cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat 3361 aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc 3421 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt 3481 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat 3541 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg 3601 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg 3661 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg 3721 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc 3781 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt 3841 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc 3901 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca 3961 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat 4021 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc 4081 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct 4141 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt 4201 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac 4261 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga 4321 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg 4381 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag 4441 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga 4501 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca 4561 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg 4621 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt 4681 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc 4741 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga 4801 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt 4861 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt 4921 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg 4981 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac 5041 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg 5101 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca 5161 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc 5221 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag 5281 aaataatttt gtttaacttt aagaaggaga tataccatgg gcagcagcca tcatcatcat 5341 catcacagca gcggcctggt gccgcgcggc agccatagcc atatgaccga cgaaggcgaa 5401 aaaccgctgg cgggtatgga ctttgcgatc gctgatagcg ttaccgacga gatgctggca 5461 gataaagcag cgctagtagt tgaagttgtg ggcgataact accatcatgc gccgatcgtg 5521 ggtatcgctt tggcgaacga gcgcggccgt tttttcctgc gcccggaaac cgcactggcg 5581 gacccgaagt ttctggcttg gctgggcgac gaaacgaaaa aaaagactat gttcgattct 5641 aaacgtgcag cggtggctct caaatggaaa ggtatcgaat tgcgcggtgt tgtgttcgac 5701 ctgctgctgg ctgcgtacct gctcgatcct gctcaggctg cgggtgacgt ggcagctgta 5761 gcgaaaatgc accagtacga agccgtacgc tctgatgaag cggtttatgg taaaggtgcg 5821 aaacgtaccg tgccagacga accaaccctg gcggaacacc tggctcgcaa agcagccgct 5881 atctgggccc tggaggagcc tctgatggat gaacttcgcc gcaacgaaca ggaccgtctg 5941 ctcacagaac tggaacagcc gttggcgggt attctggcaa acatggagtt cactggtgta 6001 aaagtggaca ctaagcgcct ggaacaaatg ggtgccgaac tgactgagca gcttcaggca 6061 gttgaacgcc gcatctacga actggcgggt caggagttta acatcaactc tcctaaacag 6121 ctgggcaccg tgttgttcga caaactgcaa ctgccggtgt tgaagaaaac gaaaaccggc 6181 tacagcacct ccgccgatgt tctggaaaaa ctggcaccgc atcacgaaat cgttgaacac 6241 atcctgcact accgtcagct gggtaaactg caatctacct acatcgaggg cttactgaaa 6301 gtagtgcacc cggtgaccgg taaagtgcac actatgttca accaggctct gacgcagacc 6361 ggccgcctgt cctccgttga accgaacctg caaaacatcc ccatccgtct ggaagagggc 6421 cgtaagatcc gtcaggcatt cgtgccgtct gaaccggact ggctgatttt cgctgctgat 6481 tactctcaaa tcgaactgcg tgttcttgct cacattgcgg aggacgataa cctgattgaa 6541 gcattccgtc gtggtctgga cattcacact aaaacggcta tggacatttt ccatgtttcc 6601 gaagaagatg tgaccgctaa tatgcgtcgt caggctaaag cggtaaactt tggtatcgta 6661 tacggtatct ccgattacgg cctggcacag aacctgaaca tcacccgcaa agaagcggct 6721 gagtttatcg aacgttattt cgcctcgttc cccggtgtta aacagtacat ggataacatc 6781 gttcaagaag ctaaacagaa gggttatgtg accaccctgc tgcaccgtcg tcgttacctg 6841 ccggacatca cctcccgtaa cttcaacgtg cgttctttcg ccgaacgtac ggctatgaat 6901 acccctatcc agggcagcgc ggcggacatc atcaaaaaag ccatgatcga cctgagcgtg 6961 cgcctgcgcg aagaacgttt gcaggctcgc ctgcttctgc aagtgcacga tgagctgatc 7021 ctagaagctc caaaagagga aattgaacgt ttgtgccgtc tggttccgga agttatggaa 7081 caggctgtcg cactgcgtgt tccgcttaaa gttgattacc attacggtcc cacttggtac 7141 gacgccaagt gaggatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc 7201 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt 7261 tttgctgaaa ggaggaacta tatccggata tcccgcaaga ggcccggcag taccggcata 7321 accaagccta tgcctacagc atccagggtg acggtgccga ggatgacgat gagcgcattg 7381 ttagatttca tacacggtgc ctgactgcgt tagcaattta actgtgataa actaccgcat 7441 taaagcttat cgatgataag ctgtcaaaca tgagaa //