LOCUS Exported 6480 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Bacterial expression for structure determination; may not be full ORF. ACCESSION . VERSION . KEYWORDS IQSEC2 (IQSEC2A-c001) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6480) TITLE SGC Oxford April 2018 plasmids REFERENCE 2 (bases 1 to 6480) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6480 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 1..20 /label=T7 /note="T7 promoter, forward primer" promoter 1..19 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 20..44 /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)." RBS 59..81 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 92..121 /codon_start=1 /product="10xHis affinity tag" /label=10xHis /translation="HHHHHHHHHH" CDS 146..166 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 1345..1362 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(1415..1433) /label=T7 Term /note="T7 terminator, reverse primer" terminator 1429..1476 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 1513..1968 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1600..1619) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1810..1831 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(2060..2875) /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind 2783..2802 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 2867..2886 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 2997..3585 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3486..3505 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3739..3756 /label=L4440 /note="L4440 vector, forward primer" misc_feature 3771..3910 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(3836..3855) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3996..4018 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(4012..4203) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(5012..6094) /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 6056..6075 /label=LacI-R /note="LacI, reverse primer" promoter complement(6095..6172) /gene="lacI" /label=lacI promoter primer_bind 6378..6397 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" ORIGIN 1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt 61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatc atcatcatca 121 ttcttctggt gtagatctgg gtaccgagaa cctgtacttc caatccatgt gcaaacagac 181 ctatcagcgt gaaacccgtc atagctggga tagtccggca tttaacaatg atgttgttca 241 gcgtcgtcat tatcgtattg gtctgaacct gtttaacaag aaaccggaaa aaggcatcca 301 gtatctgatt gaacgtggtt ttctgagcga tacaccggtt ggtgttgcac attttattct 361 ggaacgtaaa ggtctgagcc gtcagatgat tggtgaattt ctgggtaatc gtcagaaaca 421 gtttaatcgt gatgttctgg attgcgtggt tgatgaaatg gattttagca gcatggatct 481 ggatgatgca ctgcgtaaat ttcagagcca tattcgtgtt cagggtgaag cacagaaagt 541 tgaacgcctg attgaagcat ttagccagcg ttattgtgtt tgtaatccgg cactggttcg 601 tcagtttcgc aatccggata ccatttttat cctggccttt gcaattattc tgctgaacac 661 cgatatgtat agcccgagcg ttaaagcaga acgtaaaatg aaactggacg acttcattaa 721 aaacctgcgt ggtgttgata acggtgaaga tattccgcgt gatctgctgg ttggtattta 781 tcagcgtatt cagggtcgtg aactgcgtac caatgatgat catgttagcc aggttcaggc 841 agttgaacgt atgattgttg gtaaaaaacc ggttctgagc ctgccgcatc gtcgtctggt 901 ttgttgttgt cagctgtatg aagttccgga tccgaatcgt ccgcagcgtc tgggtctgca 961 tcagcgcgaa gtttttctgt ttaatgattt actggtggtg accaaaatct tccagaaaaa 1021 aaagatcctg gtgacctata gctttcgtca gagctttccg ctggttgaaa tgcacatgca 1081 gctgtttcag aacagctatt atcagtttgg cattaaactg ctgagcgcag ttccaggtgg 1141 tgaacgcaaa gttctgatta tctttaatgc accgagcctg caggatcgtc tgcgttttac 1201 cagcgatctg cgtgaaagca ttgccgaagt tcaagagatg gaaaaatatc gtgttgaaag 1261 cgagctggaa aaacagaaat gacagtaaag gtggatacgg atccgaattc gagctccgtc 1321 gacaagcttg cggccgcact cgagcaccac caccaccacc actgagatcc ggctgctaac 1381 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc 1441 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga 1501 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 1561 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 1621 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 1681 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 1741 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 1801 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 1861 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 1921 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt 1981 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 2041 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 2101 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 2161 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 2221 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 2281 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 2341 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 2401 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 2461 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 2521 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 2581 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 2641 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 2701 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 2761 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 2821 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 2881 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 2941 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 3001 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 3061 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 3121 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 3181 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 3241 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 3301 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 3361 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 3421 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 3481 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 3541 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 3601 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 3661 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 3721 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 3781 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa 3841 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt 3901 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct 3961 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt 4021 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg 4081 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag 4141 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt 4201 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg 4261 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg 4321 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca 4381 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc 4441 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga 4501 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca 4561 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg 4621 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat 4681 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc 4741 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct 4801 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag 4861 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg 4921 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg 4981 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 5041 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 5101 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg 5161 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat 5221 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc 5281 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc 5341 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca 5401 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg 5461 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga 5521 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct 5581 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt 5641 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat 5701 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt 5761 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg 5821 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg 5881 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca 5941 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg 6001 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat 6061 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt 6121 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct 6181 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg 6241 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg 6301 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga 6361 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg 6421 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat //