LOCUS Exported 7758 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pBD-0001 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7758) AUTHORS DeLaBarre B, Christianson JC, Kopito RR, Brunger AT TITLE Central pore residues mediate the p97/VCP activity required for ERAD. JOURNAL Mol Cell. 2006 May 19. 22(4):451-62. PUBMED 16713576 REFERENCE 2 (bases 1 to 7758) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7758 /organism="synthetic DNA construct" /mol_type="other DNA" terminator 26..73 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" primer_bind 69..87 /label=T7 Term /note="T7 terminator, reverse primer" CDS complement(140..157) /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS complement(2636..2653) /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS complement(2663..2680) /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" RBS 2699..2721 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" protein_bind 2736..2760 /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 complement(2760..2779) /label=T7 /note="T7 promoter, forward primer" promoter complement(2761..2779) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2863..2882) /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" promoter 3088..3165 /gene="lacI" /label=lacI promoter CDS 3166..4248 /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(3185..3204) /label=LacI-R /note="LacI, reverse primer" CDS 5057..5248 /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" primer_bind complement(5242..5264) /label=pGEX 3' /note="pGEX vectors, reverse primer" misc_feature 5350..5489 /label=bom /note="basis of mobility region from pBR322" primer_bind 5405..5424 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind complement(5504..5521) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5675..6263) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5755..5774) /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind complement(6374..6393) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 6385..7200 /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 complement(6458..6477) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin complement(7292..7747) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(7429..7450) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 7641..7660 /label=F1ori-R /note="F1 origin, reverse primer" ORIGIN 1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa 61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt 121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt 181 cgacggagct cgaattcgga tcccgtcact attagccata caggtcatcg tcattgtctt 241 ctgtgtacac actgccacct gtgccacctc cactgccctg gctgggacca gctccaccct 301 ggttccctga agggaatctg aagctgccaa aacctcgact ctgctgcagt gtctgggcga 361 acatttcata cttccgaatg tcattatcgc tgacagaacg tcgggcaaaa cgcatggctt 421 cctcaaagtg atctctgcgg atctcaggca ctggatcgtc ctcttctacc tccatagccg 481 atggatttgt ctgcctctct cgttctcgcc taatctcact ctcaatagat tcacgaatgg 541 ccagtttaca agcccgttgg caaatttctg tcaaatcagc tccagaaaag ccattagtca 601 tcttagccag gaactccaaa tccacatcct tggcaactgg ggactttcgc agattggctt 661 ttaggatggc aacacgggac ttctcatcag gaagtgggat ataaatgagc tgatctagac 721 ggccaggtct taggatagca ggatcaatga tgtcaggcct gttggtagct ccaatgataa 781 acacattctt ttttgtagac atgccatcca tttctgtcaa gatctgattg atgactcggt 841 cagcagctcc accaccatct ccaatattac caccacgagc cttggcaatt gaatctaact 901 catcaaagaa gagtacacag ggggcagctt gccgtgcctt gtcaaaaatt tcccggacat 961 tggcctcaga ttccccaaac cacatggtaa gcagctcagg acccttgatg gagatgaagt 1021 tggcctggca ttcattagca atggctttag ccagtaaggt tttcccacag ccaggaggtc 1081 catagaaaag aacacctttg gagggagtca tgccaaattt gaggaatttg tctggatgtt 1141 ccacggggta ctgaaccaac tcctgaagct cacgtttgac atcctccagg cctccaatat 1201 cttcccaggt tacttgtggc acctctacca cagtttcccg aagtgctgat gggttgcttt 1261 gactcaaagc ccaccggaag tcatccatag taactgccag ggaattcatg acctcagcat 1321 caatggtctc atcttctagg tcaatgaggt ccattttttt ccggatggcc tgcagagcag 1381 cctctgaaca tagggctgcc aaatcagcac caacatgacc atgagtctca ttggctacct 1441 gttccaagtc cacatcatct gccagtttca tgttcttggt atggatctga agaatctcca 1501 aacgtcctgt agcatcaggt attccaatat ctacctctct gtcaaagcga ccaaatcgcc 1561 gtagggctgg gtcaatgctg ttgggtctat tggttgctgc cataactatc acatgtgctc 1621 tctgctttag gccatccatg agggtcaaca actgagacac gatgcgacgc tccacttccc 1681 catgagtttt ctctcttttg ggtgcaatgg catcaagctc atcgatgaag atgatagcag 1741 gagcattctt ttcagcttcc tcaaaggctt tacgaaggtt gctctcagac tcaccagcca 1801 atttgctcat gatttcagga ccattgatca gaaagaagaa ggctccagtt tcatttgcca 1861 cagctcgagc aatcagggtc ttccctgtcc caggaggccc atacaacaag attccccgag 1921 gaggctttac accaatcgcc ttgaagagcg caggatgtct cagtggcagc tccaccatct 1981 cctttatctg agctagctgc ttcctgcaac caccgacgtc atcatagcct acttcattca 2041 aggattcctc ctcatcctct cgtttgattg gctccccctc acagtggatc actgtgtctg 2101 gagcaacaat acagtaaggg ctgggatctg tctctacaac tttgaactcc acagcacgca 2161 tcccaccccg gacaagaaaa atatctcctt tacggatggg ccgataagct tccaggaagt 2221 acggcttaag gtatacctca aagagattgc cagtgatgcc ttccactgtg tcatcgatgg 2281 gtagaacgtg gatacgtttg ccatacttta catcagggca tggctggatg ctgatgacat 2341 ctcctaggcg aactcggagg ttattccgaa caactctatt cattcgaatc ttctcatcag 2401 aacacgtgtc atcagaaaga acaatgcata cagcttcccg tcttttcttt ccttttagca 2461 acaccgtgtc acctcggaac aactgcagtt catccatctt gggctgggac aaggacacca 2521 cgctgttatc ttcattgatg gcttcatcaa caattaaccg attgggtcgg ttcttctgtt 2581 tgagaatggc tgttgataaa tcatcacctt ttgaatcggc tccagaggcc atatggctgc 2641 cgcgcggcac caggccgctg ctgtgatgat gatgatgatg gctgctgccc atggtatatc 2701 tccttcttaa agttaaacaa aattatttct agaggggaat tgttatccgc tcacaattcc 2761 cctatagtga gtcgtattaa tttcgcggga tcgagatctc gatcctctac gccggacgca 2821 tcgtggccgg catcaccggc gccacaggtg cggttgctgg cgcctatatc gccgacatca 2881 ccgatgggga agatcgggct cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta 2941 tggtggcagg ccccgtggcc gggggactgt tgggcgccat ctccttgcat gcaccattcc 3001 ttgcggcggc ggtgctcaac ggcctcaacc tactactggg ctgcttccta atgcaggagt 3061 cgcataaggg agagcgtcga gatcccggac accatcgaat ggcgcaaaac ctttcgcggt 3121 atggcatgat agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg 3181 ttatacgatg tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac 3241 caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg 3301 aattacattc ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc 3361 gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct 3421 cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa 3481 gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac 3541 tatccgctgg atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg 3601 ttatttcttg atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac 3661 ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta 3721 gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc 3781 actcgcaatc aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt 3841 tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc 3901 aacgatcaga tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt 3961 gcggatatct cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg 4021 ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg 4081 caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa 4141 agaaaaacca ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca 4201 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat 4261 taatgtaagt tagctcactc attaggcacc gggatctcga ccgatgccct tgagagcctt 4321 caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac 4381 tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgctctggg tcattttcgg 4441 cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat 4501 cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa 4561 gcaggccatt atcgccggca tggcggcccc acgggtgcgc atgatcgtgc tcctgtcgtt 4621 gaggacccgg ctaggctggc ggggttgcct tactggttag cagaatgaat caccgatacg 4681 cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa caacatgaat 4741 ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat 4801 tatgttccgg atctgcatcg caggatgctg ctggctaccc tgtggaacac ctacatctgt 4861 attaacgaag cgctggcatt gaccctgagt gatttttctc tggtcccgcc gcatccatac 4921 cgccagttgt ttaccctcac aacgttccag taaccgggca tgttcatcat cagtaacccg 4981 tatcgtgagc atcctctctc gtttcatcgg tatcattacc cccatgaaca gaaatccccc 5041 ttacacggag gcatcagtga ccaaacagga aaaaaccgcc cttaacatgg cccgctttat 5101 cagaagccag acattaacgc ttctggagaa actcaacgag ctggacgcgg atgaacaggc 5161 agacatctgt gaatcgcttc acgaccacgc tgatgagctt taccgcagct gcctcgcgcg 5221 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg 5281 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg 5341 gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac 5401 tatgcggcat cagagcagat tgtactgaga gtgcaccatt gcggtgtgaa ataccgcaca 5461 gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc 5521 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 5581 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 5641 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 5701 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 5761 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 5821 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 5881 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 5941 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 6001 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 6061 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag 6121 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 6181 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 6241 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 6301 agtggaacga aaactcacgt taagggattt tggtcatgaa caataaaact gtctgcttac 6361 ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc ttgctctagg 6421 ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat 6481 gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc gccagagttg 6541 tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta 6601 aactggctga cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat 6661 gatgcatggt tactcaccac tgcgatcccc gggaaaacag cattccaggt attagaagaa 6721 tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat 6781 tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg 6841 caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc 6901 tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca 6961 gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata 7021 ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta 7081 tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt 7141 attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa 7201 gaattaattc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt 7261 tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa 7321 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa 7381 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac 7441 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag 7501 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt 7561 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg 7621 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca 7681 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag 7741 ggcgcgtccc attcgcca //