LOCUS Exported 6775 bp ds-DNA circular SYN 29-JUL-2022 DEFINITION Codon optimized (E. coli) TEVp fused with GST and his-tagged for purification. ACCESSION . VERSION . KEYWORDS pET22b-6h-GST-TEVp SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6775) AUTHORS Ahan RE, Kirpat BM, Saltepe B, Seker UOS TITLE A Self-Actuated Cellular Protein Delivery Machine. JOURNAL ACS Synth Biol. 2019 Apr 19;8(4):686-696. doi: 10.1021/acssynbio.9b00062. Epub 2019 Mar 12. PUBMED 30811932 REFERENCE 2 (bases 1 to 6775) AUTHORS . TITLE Direct Submission JOURNAL Exported Jul 29, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6775 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 86..163 /gene="lacI" /label=lacI promoter CDS 164..1246 /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(183..202) /label=LacI-R /note="LacI, reverse primer" CDS 2055..2246 /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" primer_bind complement(2240..2262) /label=pGEX 3' /note="pGEX vectors, reverse primer" misc_feature 2348..2487 /label=bom /note="basis of mobility region from pBR322" primer_bind 2403..2422 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind complement(2502..2519) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2673..3261) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2753..2772) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3432..4292) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 4055..4074 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4293..4397) /gene="bla" /label=AmpR promoter rep_origin complement(4423..4878) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4560..4581) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 4772..4791 /label=F1ori-R /note="F1 origin, reverse primer" terminator 4915..4962 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" primer_bind 4958..4976 /label=T7 Term /note="T7 terminator, reverse primer" CDS complement(5029..5046) /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS complement(5062..5769) /codon_start=1 /product="tobacco etch virus protease (Kapust et al., 2001)" /label=TEV protease /translation="GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNK HLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQ REERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFI VGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPE EPFQPVKEATQLMN" CDS complement(5785..6438) /codon_start=1 /product="glutathione S-transferase from Schistosoma japonicum" /label=GST /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK" primer_bind complement(5805..5827) /label=pGEX 5' /note="pGEX vectors, Glutathione-S-transferase, forward primer" CDS complement(6448..6465) /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" protein_bind 6509..6533 /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(6533..6552) /label=T7 /note="T7 promoter, forward primer" promoter complement(6534..6552) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(6636..6655) /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" ORIGIN 1 gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg 61 cataagggag agcgtcgaga tcccggacac catcgaatgg cgcaaaacct ttcgcggtat 121 ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt 181 atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca 241 ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa 301 ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt 361 tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg 421 cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc 481 ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta 541 tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt 601 atttcttgat gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg 661 tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc 721 gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac 781 tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt 841 tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa 901 cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc 961 ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt 1021 aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca 1081 actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag 1141 aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt 1201 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta 1261 atgtaagtta gctcactcat taggcaccgg gatctcgacc gatgcccttg agagccttca 1321 acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca cttatgactg 1381 tcttctttat catgcaactc gtaggacagg tgccggcagc gctctgggtc attttcggcg 1441 aggaccgctt tcgctggagc gcgacgatga tcggcctgtc gcttgcggta ttcggaatct 1501 tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc 1561 aggccattat cgccggcatg gcggccccac gggtgcgcat gatcgtgctc ctgtcgttga 1621 ggacccggct aggctggcgg ggttgcctta ctggttagca gaatgaatca ccgatacgcg 1681 agcgaacgtg aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg 1741 tcttcggttt ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta 1801 tgttccggat ctgcatcgca ggatgctgct ggctaccctg tggaacacct acatctgtat 1861 taacgaagcg ctggcattga ccctgagtga tttttctctg gtcccgccgc atccataccg 1921 ccagttgttt accctcacaa cgttccagta accgggcatg ttcatcatca gtaacccgta 1981 tcgtgagcat cctctctcgt ttcatcggta tcattacccc catgaacaga aatccccctt 2041 acacggaggc atcagtgacc aaacaggaaa aaaccgccct taacatggcc cgctttatca 2101 gaagccagac attaacgctt ctggagaaac tcaacgagct ggacgcggat gaacaggcag 2161 acatctgtga atcgcttcac gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt 2221 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc 2281 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt 2341 gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta 2401 tgcggcatca gagcagattg tactgagagt gcaccattgc ggtgtgaaat accgcacaga 2461 tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg 2521 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 2581 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 2641 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 2701 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 2761 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 2821 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 2881 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 2941 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 3001 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 3061 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 3121 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 3181 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 3241 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 3301 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 3361 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 3421 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 3481 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 3541 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 3601 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 3661 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 3721 agtttgcgca acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt 3781 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 3841 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 3901 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 3961 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 4021 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 4081 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 4141 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 4201 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 4261 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 4321 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 4381 caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata 4441 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg 4501 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc 4561 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa 4621 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt 4681 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac 4741 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta 4801 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg 4861 cgccgctaca gggcgcgtcc cattcgccaa tccggatata gttcctcctt tcagcaaaaa 4921 acccctcaag acccgtttag aggccccaag gggttatgct agttattgct cagcggtggc 4981 agcagccaac tcagcttcct ttcgggcttt gttagcagcc ggatctcagt ggtggtggtg 5041 gtggtgctcg agtcatcatt cattcatcag ctgggtggct tctttcaccg gctggaacgg 5101 ttcttccggt ttcaccatga acactttatg gccgccccac agcacgctat ctgcattcag 5161 gcgccaaccg ctcacccact gctgtgcttc ctggttggtc agcagttcca tgaagttctt 5221 cggcacgctg gtaaagtaat tattggtgtt ggtaaaatta ctggcactat gaatgcccac 5281 gataaagcca tcgcgggtac tcaccagcgg gctgccacac tggccatctt tggtttgaat 5341 ccagtgtttc cagaagatgc catcgctgct cggaaaggtg caactggtat cgctcaccat 5401 gctgctcatg cttttggtct gaaaattggt ggtcaccaga caaatgcgtt cttcgcgctg 5461 cggttcgcgg aatttcagtt tctgcggaaa cggcgggaag tctttcggca tgcggatgat 5521 aatcatgtcg cggccatcga tcaggtgctg ctgcagggtg gtggtgttct tcactttgaa 5581 cacgccatgc aggctctgca ccagcagggt gccattgttg cggcgaaaca ggtgtttgtt 5641 ggtgatgata aacgggccga agccgatgcc atacaggctg gtggtatggc catcactctc 5701 gttggtcagg tggcagatgg tactgctgat cgggttgtaa tcgcgaggac ccttaaacag 5761 gctctcaccc gggcctttgg atccttttgg aggatggtcg ccaccaccaa acgtggcttg 5821 ccagccctgc aaaggccatg ctatatactt gctggatttc aagtacttat caatttgtgg 5881 gatagcttca atacgttttt taaaacaaac taattttggg aacgcatcca ggcacattgg 5941 gtccatgtat aaaacaacat caagagcgtc atacaacatg aagtcaggat gggttacatg 6001 atcaccattt aaatatgttt tatgacataa acgatcttcg aacattttca gcatttcagg 6061 tagcttgcta agaaaatcaa ctttgagagt ttcaaagtct ttactatatg caattctcga 6121 aacaccgtat ctaatatcca aaaccgctcc ttcaagcatt gaaatctctg cacgctcttt 6181 tggacaacca cccaacatgt tgtgcttgtc agctatataa cgtatgatgg ccatagactg 6241 tgttaattta acatcaccat caatataata aggaagattg ggaaactcca aacccaattc 6301 aaactttttg tttcgccatt tatcaccttc atcgcgctca tacaaatgct cttcatattt 6361 ttcttcaaga tattccaaaa gaagtcgagt gggttgcaca aggcccttaa ttttccaata 6421 acctagtata ggggacatac tagtgttgtg atggtgatgg tgatgaatgg tgttcatggt 6481 accctccttc ttaaagttaa tctagagggg aattgttatc cgctcacaat tcccctatag 6541 tgagtcgtat taatttcgcg ggatcgagat ctcgatcctc tacgccggac gcatcgtggc 6601 cggcatcacc ggcgccacag gtgcggttgc tggcgcctat atcgccgaca tcaccgatgg 6661 ggaagatcgg gctcgccact tcgggctcat gagcgcttgt ttcggcgtgg gtatggtggc 6721 aggccccgtg gccgggggac tgttgggcgc catctccttg catgcaccat tcctt //