LOCUS 40924_790 6256 bp DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression vector encoding for the ER targeted and FLAGM1 tagged redox probe roGFPiE (roGFP C147, E147b, C204) . ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6256) AUTHORS Avezov E, Cross BC, Kaminski Schierle GS, Winters M, Harding HP, Melo EP, Kaminski CF, Ron D TITLE Lifetime imaging of a fluorescent protein sensor reveals surprising stability of ER thiol redox. JOURNAL J Cell Biol. 2013 Apr 15;201(2):337-49. doi: 10.1083/jcb.201211155. PUBMED 23589496 REFERENCE 2 (bases 1 to 6256) TITLE Direct Submission REFERENCE 3 (bases 1 to 6256) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; doi: "10.1083/jcb"; journalName: "J Cell Biol"; date: "2013-04-15- 15"; volume: "201"; issue: "2"; pages: "337-49" COMMENT SGRef: number: 2; type: "Journal Article" FEATURES Location/Qualifiers source 1..6256 /mol_type="other DNA" /organism="synthetic DNA construct" primer_bind complement(44..63) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 235..614 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 615..818 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 815..839 /label=LNCX /note="Human CMV promoter, forward primer" CDS 949..972 /codon_start=1 /label=FLAG /note="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /translation="DYKDDDDK" primer_bind complement(1006..1034) /label=GFP-R /note="GFP, reverse primer. Does NOT anneal to EGFP" primer_bind 1632..1652 /label=GFP-F /note="GFP, forward primer. Does NOT anneal to EGFP" promoter complement(1808..1826) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" polyA_signal 1852..2076 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2122..2550 /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter 2564..2894 /label=SV40 promoter /note="SV40 enhancer and early promoter" CDS 2961..3752 /codon_start=1 /label=NeoR/KanR /note="aminoglycoside phosphotransferase" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" polyA_signal 3929..4062 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4099..4115) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind complement(4123..4139) /label=lac operator /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)." promoter complement(4147..4177) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind complement(4192..4213) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4330..4347) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4501..5089) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(5263..6120) /codon_start=1 /label=AmpR /note="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(6121..6225) /label=AmpR promoter ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 781 gtaggcgtgt acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg 841 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgactc tagaggatcc 901 gccatgagcg ccctgctgat cctggctctc gtgggcgctg ccgtggccga ctacaaggac 961 gacgacgaca agtccggaag taaaggagaa gaacttttca ctggagttgt cccaattctt 1021 gttgaattag atggtgatgt taatgggcac aaattttctg tcagtggaga gggtgaaggt 1081 gatgcaacat acggaaaact tacccttaaa tttatttcca ctactggaaa actacctgtt 1141 ccatggccaa cacttgtcac tactctgagt tatggtgttc aatgcttttc aagataccca 1201 gatcatatga aacggcatga ctttttcaag agtgccatgc ccgaaggtta tgtacaggaa 1261 agaactataa gcttcaaaga tgacgggaac tacaagacac gtgctgaagt caagtttgaa 1321 ggtgataccc ttgttaatag aatcgagtta aaaggtattg attttaaaga agatggaaac 1381 attcttggac acaaattgga atacaactat aactgcgagt ccaatgtata catcatggca 1441 gacaaacaaa agaatggaat caaagcgaac ttcaaaacca gacacaacat tgaagatgga 1501 agcgttcaac tagcagacca ttatcaacaa aatactccaa ttggcgatgg ccctgtcctt 1561 ttaccagaca accattacct gtccacatgc tctgcccttt cgaaagatcc caacgaaaag 1621 agagaccaca tggtccttct tgagtttgta acagctgctg ggattacaca tggcatggat 1681 gaactataca aagatgagct ataaggtacc gagctcggat ccactagtaa cggccgccag 1741 tgtgctggaa ttctgcagat atccatcaca ctggcggccg ctcgagcatg catctagagg 1801 gccctattct atagtgtcac ctaaatgcta gagctcgctg atcagcctcg actgtgcctt 1861 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg 1921 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt 1981 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca 2041 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct 2101 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg 2161 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt 2221 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcggggca 2281 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg 2341 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg 2401 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct 2461 cggtctattc ttttgattta taagggattt tggggatttc ggcctattgg ttaaaaaatg 2521 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg 2581 tggaaagtcc ccaggctccc caggcaggca gaagtatgca aagcatgcat ctcaattagt 2641 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc 2701 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc 2761 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg 2821 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc 2881 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga 2941 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg 3001 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg 3061 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt 3121 ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg 3181 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat 3241 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat 3301 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg 3361 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg 3421 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc 3481 tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc 3541 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg 3601 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg 3661 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca 3721 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac 3781 cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga 3841 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga 3901 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa 3961 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg 4021 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta 4081 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat 4141 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag 4201 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg 4261 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc 4321 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc 4381 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa 4441 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 4501 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 4561 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 4621 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 4681 cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 4741 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 4801 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 4861 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 4921 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac 4981 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 5041 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 5101 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 5161 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 5221 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 5281 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 5341 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 5401 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 5461 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 5521 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 5581 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 5641 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 5701 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 5761 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac 5821 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg 5881 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc 5941 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg 6001 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac 6061 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat 6121 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 6181 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 6241 agtgccacct gacgtc //