LOCUS Exported 7489 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Bacterial expression of His-tagged KPNB1 in the pET28 protein expression backbone. ACCESSION . VERSION . KEYWORDS pET28-KPNB1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7489) TITLE Simon lab plasmids REFERENCE 2 (bases 1 to 7489) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7489 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 45..67 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(61..252) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(1061..2143) /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 2105..2124 /label=LacI-R /note="LacI, reverse primer" promoter complement(2144..2221) /gene="lacI" /label=lacI promoter primer_bind 2427..2446 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 2530..2549 /label=T7 /note="T7 promoter, forward primer" promoter 2530..2548 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 2549..2573 /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 2588..2610 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 2629..2646 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 2656..2673 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS 4879..4896 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(4949..4967) /label=T7 Term /note="T7 terminator, reverse primer" terminator 4963..5010 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 5047..5502 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5134..5153) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5344..5365 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(5595..6410) /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 6318..6337 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 6402..6421 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 6532..7120 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7021..7040 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 7274..7291 /label=L4440 /note="L4440 vector, forward primer" misc_feature 7306..7448 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(7374..7393) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg 61 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc 121 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt 181 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc 241 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc 301 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact 361 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac 421 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca 481 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag 541 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt 601 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag 661 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg 721 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt 781 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat 841 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg 901 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg 961 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg 1021 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc 1081 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt 1141 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc 1201 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca 1261 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat 1321 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc 1381 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct 1441 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt 1501 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac 1561 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga 1621 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg 1681 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag 1741 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga 1801 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca 1861 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg 1921 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt 1981 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc 2041 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga 2101 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt 2161 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt 2221 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg 2281 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac 2341 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg 2401 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca 2461 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc 2521 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag 2581 aaataatttt gtttaacttt aagaaggaga tataccatgg gcagcagcca tcatcatcat 2641 catcacagca gcggcctggt gccgcgcggc agccatatga aggagtcgac attggaagcc 2701 atcggttata tttgccaaga tatagaccca gagcagctac aagataaatc caatgagatt 2761 ctgactgcca taatccaggg gatgaggaaa gaagagccta gtaataatgt gaagctagct 2821 gctacgaatg cactcctgaa ctcattggag ttcaccaaag caaactttga taaagagtct 2881 gaaaggcact ttattatgca ggtggtctgt gaagccacac agtgtccaga tacgagggta 2941 cgagtggctg ctttacagaa tctggtgaag ataatgtcct tatattatca gtacatggag 3001 acatatatgg gtcctgctct ttttgcaatc acaatcgaag caatgaaaag tgacattgat 3061 gaggtggctt tacaagggat agaattctgg tccaatgtct gtgatgagga aatggatttg 3121 gccattgaag cttcagaggc agcagaacaa ggacggcccc ctgagcacac cagcaagttt 3181 tatgcgaagg gagcactaca gtatctggtt ccaatcctca cacagacact aactaaacag 3241 gacgaaaatg atgatgacga tgactggaac ccctgcaaag cagcaggggt gtgcctcatg 3301 cttctggcca cctgctgtga agatgacatt gtcccacatg tcctcccctt cattaaagaa 3361 cacatcaaga acccagattg gcggtaccgg gatgcagcag tgatggcttt tggttgtatc 3421 ttggaaggac cagagcccag tcagctcaaa ccactagtta tacaggctat gcccacccta 3481 atagaattaa tgaaagaccc cagtgtagtt gttcgagata cagctgcatg gactgtaggc 3541 agaatttgtg agctgcttcc tgaagctgcc atcaatgatg tctacttggc tcccctgcta 3601 cagtgtctga ttgagggtct cagtgctgaa cccagagtgg cttcaaatgt gtgctgggct 3661 ttctccagtc tggctgaagc tgcttatgaa gctgcagacg ttgctgatga tcaggaagaa 3721 ccagctactt actgcttatc ttcttcattt gaactcatag ttcagaagct cctagagact 3781 acagacagac ctgatggaca ccagaacaac ctgaggagtt ctgcatatga atctctgatg 3841 gaaattgtga aaaacagtgc caaggattgt tatcctgctg tccagaaaac gactttggtc 3901 atcatggaac gactgcaaca ggttcttcag atggagtcac atatccagag cacatccgat 3961 agaatccagt tcaatgacct tcagtcttta ctctgtgcaa ctcttcagaa tgttcttcgg 4021 aaagtgcaac atcaagatgc tttgcagatc tctgatgtgg ttatggcctc cctgttaagg 4081 atgttccaaa gcacagctgg gtctggggga gtacaagagg atgccctgat ggcagttagc 4141 acactggtgg aagtgttggg tggtgaattc ctcaagtaca tggaggcctt taaacccttc 4201 ctgggcattg gattaaaaaa ttatgctgaa taccaggttt gtttggcagc tgtgggctta 4261 gtgggagact tgtgccgtgc cctgcaatcc aacatcatac ctttctgtga cgaggtgatg 4321 cagctgcttc tggaaaattt ggggaatgag aacgtccaca ggtctgtgaa gccgcagatt 4381 ctgtcagtgt ttggtgatat tgcccttgct attggaggag agtttaaaaa atacttagag 4441 gttgtattga atactcttca gcaggcctcc caagcccagg tggacaagtc agactatgac 4501 atggtggatt atctgaatga gctaagggaa agctgcttgg aagcctatac tggaatcgtc 4561 cagggattaa agggggatca ggagaacgta cacccggatg tgatgctggt acaacccaga 4621 gtagaattta ttctgtcttt cattgaccac attgctggag atgaggatca cacagatgga 4681 gtagtagctt gtgctgctgg actaataggg gacttatgta cagcatttgg gaaggatgta 4741 ctgaaattag tagaagctag gccaatgatc catgaattgt taactgaagg gcggagatcg 4801 aagactaaca aagcaaaaac ccttgctaca tgggcaacaa aagaactgag gaaactgaag 4861 aaccaagctt gactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc 4921 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg 4981 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg 5041 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg 5101 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc 5161 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc 5221 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta 5281 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta 5341 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg 5401 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa 5461 aatttaacgc gaattttaac aaaatattaa cgtttacaat ttcaggtggc acttttcggg 5521 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 5581 tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata 5641 tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca 5701 ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca 5761 acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca 5821 ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact 5881 tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta 5941 ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta 6001 caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca 6061 cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg 6121 agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat 6181 tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg 6241 ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca 6301 cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg 6361 gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt 6421 gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga 6481 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 6541 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 6601 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 6661 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc 6721 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 6781 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 6841 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 6901 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 6961 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 7021 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 7081 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt 7141 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc 7201 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg 7261 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt 7321 tctccttacg catctgtgcg gtatttcaca ccgcatatat ggtgcactct cagtacaatc 7381 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca 7441 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggc //