LOCUS Exported 5758 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Bacterial expression for structure determination; may not be full ORF. ACCESSION . VERSION . KEYWORDS CBS SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5758) TITLE SGC Oxford REFERENCE 2 (bases 1 to 5758) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5758 /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..109 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 134..154 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 619..636 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(689..707) /label=T7 Term /note="T7 terminator, reverse primer" terminator 703..750 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 787..1242 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(874..893) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1084..1105 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(1335..2150) /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 2058..2077 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 2142..2161 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 2272..2860 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2761..2780 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3014..3031 /label=L4440 /note="L4440 vector, forward primer" misc_feature 3046..3188 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(3114..3133) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3274..3296 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(3290..3481) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(4290..5372) /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 5334..5353 /label=LacI-R /note="LacI, reverse primer" promoter complement(5373..5450) /gene="lacI" /label=lacI promoter primer_bind 5656..5675 /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 catcatcatt cttctggtgt 121 agatctgggt accgagaacc tgtacttcca atccatgaag ccctggtggt ggcacctccg 181 tgttcaggag ctgggcctgt cagccccgct gaccgtgctc ccgaccatca cctgtgggca 241 caccatcgag atcctccggg agaagggctt cgaccaggcg cccgtggtgg atgaggcggg 301 ggtaatcctg ggaatggtga cgcttgggaa catgctctcg tccctgcttg ccgggaaggt 361 gcagccgtca gaccaagttg gcaaagtcat ctacaagcag ttcaaacaga tccgcctcac 421 ggacacgctg ggcaggctct cgcacatcct ggagatggac cacttcgccc tggtggtgca 481 cgagcagcag cggcagatgg tgttcggggt ggtcaccgcc attgacttgc tgaacttcgt 541 ggccgcccag gagtgacagt aaaggtggat acggatccga attcgagctc cgtcgacaag 601 cttgcggccg cactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc 661 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg 721 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg 781 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg 841 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc 901 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc 961 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta 1021 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta 1081 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg 1141 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa 1201 aatttaacgc gaattttaac aaaatattaa cgtttacaat ttcaggtggc acttttcggg 1261 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 1321 tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata 1381 tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca 1441 ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca 1501 acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca 1561 ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact 1621 tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta 1681 ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta 1741 caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca 1801 cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg 1861 agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat 1921 tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg 1981 ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca 2041 cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg 2101 gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt 2161 gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga 2221 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 2281 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 2341 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 2401 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc 2461 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 2521 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 2581 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 2641 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 2701 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 2761 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 2821 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt 2881 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc 2941 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg 3001 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt 3061 tctccttacg catctgtgcg gtatttcaca ccgcatatat ggtgcactct cagtacaatc 3121 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca 3181 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc 3241 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt 3301 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa 3361 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg 3421 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca 3481 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag 3541 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg 3601 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat 3661 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga 3721 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa 3781 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt 3841 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc 3901 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc 3961 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt 4021 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc 4081 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt 4141 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga 4201 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag 4261 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 4321 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 4381 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc 4441 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc 4501 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc 4561 actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc 4621 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt 4681 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc 4741 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca 4801 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc 4861 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca 4921 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc 4981 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc 5041 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca 5101 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc 5161 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg 5221 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca 5281 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac 5341 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc 5401 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg 5461 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt 5521 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc 5581 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc 5641 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc 5701 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaat //