LOCUS Exported 4148 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses rsEGFP2 in bacterial cells. ACCESSION . VERSION . KEYWORDS pQE31-rsEGFP2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4148) AUTHORS Grotjohann T, Testa I, Reuss M, Brakemann T, Eggeling C, Hell SW, Jakobs S TITLE rsEGFP2 enables fast RESOLFT nanoscopy of living cells. JOURNAL elife. 2012;1:e00248. doi: 10.7554/eLife.00248. Epub 2012 Dec 31. PUBMED 23330067 REFERENCE 2 (bases 1 to 4148) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4148 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 10..54 /label=T5 promoter /note="bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator" protein_bind 30..46 /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)." protein_bind 62..78 /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 97..108 /note="strong bacterial ribosome binding site (Elowitz and Leibler, 2000)" CDS 127..144 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 154..870 /codon_start=1 /product="reversibly and rapidly switchable enhanced green fluorescent protein (Grotjohann et al., 2012)" /label=rsEGFP2 /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLAYGVLCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK SNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(199..220) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(460..479) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 807..828 /label=EGFP-C /note="EGFP, forward primer" terminator 895..989 /label=lambda t0 terminator /note="transcription terminator from phage lambda" CDS 1033..1692 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(1099..1118) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" terminator 1757..1843 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" primer_bind complement(1890..1912) /label=pGEX 3' /note="pGEX vectors, reverse primer" misc_feature 1998..2138 /label=bom /note="basis of mobility region from pBR322" primer_bind 2053..2072 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind complement(2153..2170) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2324..2912) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2404..2423) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3083..3943) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 3706..3725 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(3944..4048) /gene="bla" /label=AmpR promoter primer_bind 4116..4134 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" ORIGIN 1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca 61 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aactatgaga 121 ggatctcacc atcaccatca ccatacggat ccgatggtga gcaagggcga ggagctgttc 181 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc 241 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc 301 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctggc ctacggcgtg 361 ctgtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg 421 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc 481 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc 541 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac 601 aacgtctata tcatggccga caagcagaag aacggcatca agtctaactt caagatccgc 661 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc 721 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc caagctgagc 781 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg 841 atcactctcg gcatggacga gctgtacaag taaaagctta attagctgag cttggactcc 901 tgttgataga tccagtaatg acctcagaac tccatctgga tttgttcaga acgctcggtt 961 gccgccgggc gttttttatt ggtgagaatc caagctagct tggcgagatt ttcaggagct 1021 aaggaagcta aaatggagaa aaaaatcact ggatatacca ccgttgatat atcccaatgg 1081 catcgtaaag aacattttga ggcatttcag tcagttgctc aatgtaccta taaccagacc 1141 gttcagctgg atattacggc ctttttaaag accgtaaaga aaaataagca caagttttat 1201 ccggccttta ttcacattct tgcccgcctg atgaatgctc atccggaatt tcgtatggca 1261 atgaaagacg gtgagctggt gatatgggat agtgttcacc cttgttacac cgttttccat 1321 gagcaaactg aaacgttttc atcgctctgg agtgaatacc acgacgattt ccggcagttt 1381 ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa acctggccta tttccctaaa 1441 gggtttattg agaatatgtt tttcgtctca gccaatccct gggtgagttt caccagtttt 1501 gatttaaacg tggccaatat ggacaacttc ttcgcccccg ttttcaccat gggcaaatat 1561 tatacgcaag gcgacaaggt gctgatgccg ctggcgattc aggttcatca tgccgtttgt 1621 gatggcttcc atgtcggcag aatgcttaat gaattacaac agtactgcga tgagtggcag 1681 ggcggggcgt aattttttta aggcagttat tggtgccctt aaacgcctgg ggtaatgact 1741 ctctagcttg aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt 1801 tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccct ctagagctgc 1861 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc 1921 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt 1981 gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact 2041 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa 2101 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca 2161 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 2221 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 2281 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 2341 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 2401 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 2461 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 2521 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 2581 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 2641 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 2701 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 2761 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 2821 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 2881 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 2941 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 3001 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 3061 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 3121 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 3181 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 3241 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 3301 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 3361 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 3421 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 3481 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 3541 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 3601 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 3661 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 3721 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 3781 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 3841 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 3901 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat 3961 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 4021 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct 4081 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc 4141 gtcttcac //