LOCUS Exported 6210 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Periplasmic OM bound mNeongreen mCherry tandem, ~16 % energy transfer. pNM004 - pTHV037-OmpA-SA1-177-(SA-1)-LEDPPAEL-mNG-mCh. ACCESSION . VERSION . KEYWORDS OmpA-mNG-mCh SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6210) AUTHORS Meiresonne NY, van der Ploeg R, Hink MA, den Blaauwen T TITLE Activity-Related Conformational Changes in d,d-Carboxypeptidases Revealed by In Vivo Periplasmic Forster Resonance Energy Transfer Assay in Escherichia coli. JOURNAL MBio. 2017 Sep 12;8(5). pii: mBio.01089-17. doi: 10.1128/mBio.01089-17. PUBMED 28900026 REFERENCE 2 (bases 1 to 6210) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6210 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 53..82 /label=trc promoter /note="strong E. coli promoter; hybrid between the trp and lac UV5 promoters" protein_bind 90..106 /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 95..117 /label=M13/pUC Reverse /note="In lacZ gene" sig_peptide 133..192 /product="signal peptide from the E. coli outer membrane protein OmpA" /label=OmpA signal peptide CDS 793..1500 /codon_start=1 /product="bright monomeric yellow-green fluorescent protein derived from LanYFP (Shaner et al., 2013)" /label=mNeonGreen /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ KAFTDVMGMDELYK" CDS 1507..2217 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYK" primer_bind complement(1652..1670) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 1920..1939 /label=mCherry-F /note="mCherry, forward primer" primer_bind complement(2254..2271) /label=pBAD Reverse /note="For vectors with E. coli araBAD promoter, reverse primer" terminator 2424..2510 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 2602..2629 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" promoter 2648..2739 /gene="bla" /label=AmpR promoter CDS 2740..3600 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(2958..2977) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 3771..4359 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4260..4279 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4513..4530 /label=L4440 /note="L4440 vector, forward primer" misc_feature 4545..4685 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(4611..4630) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4771..4793 /label=pGEX 3' /note="pGEX vectors, reverse primer" promoter 4871..4948 /gene="lacI (mutant)" /label=lacIq promoter /note="In the lacIq allele, a single base change in the promoter boosts expression of the lacI gene about 10-fold." CDS 4949..6031 /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(4968..4987) /label=LacI-R /note="LacI, reverse primer" ORIGIN 1 gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc tgtttacaat 61 taatcatccg gctcgtataa tgtgtggaat tgtgagcgga taacaatttc acacaggaaa 121 cagaccatgg caaaaaagac agctatcgcg attgcagtgg cactggctgg tttcgctacc 181 gtagcgcagg ccgctccgaa agataacacc tggtacactg gtgctaaact gggctggtcc 241 cagtaccatg acactggcct catcggccaa cgcttggaga tctgccagaa cgtttgctac 301 tacctcggga cgctcaatgg tccgacccat gaaaaccaac tgggcgctgg tgcttttggt 361 ggttaccagg ttaacccgta tgttggcttt gaaatgggtt acgactggtt aggtcgtatg 421 ccgtacaaag gcagcgttga aaacggtgca tacaaagctc agggcgttca actgaccgct 481 aaactgggtt acccaatcac tgacgacctg gacatctaca ctcgtctggg tggcatggta 541 tggcgtgcag acactaaatc caacgtttat ggtaaaaacc acgacaccgg cgtttctccg 601 gtcttcgctg gcggtgttga gtacgcgatc actcctgaaa tcgctacccg tctggaatac 661 cagtggacca acaacatcgg tgacgcaggc cagatcggcc agcgtccgga caacggcatg 721 ctgagcctgg gtgtttccta ccgtttcggt cagggcgaag cagctccact tgaggatccc 781 cccgctgaat tgatggtgag caagggcgag gaggataaca tggcctctct cccagcgaca 841 catgagttac acatctttgg ctccatcaac ggtgtggact ttgacatggt gggtcagggc 901 accggcaatc caaatgatgg ttatgaggag ttaaacctga agtccaccaa gggtgacctc 961 cagttctccc cctggattct ggtccctcat atcgggtatg gcttccatca gtacctgccc 1021 taccctgacg ggatgtcgcc tttccaggcc gcgatggtag atggctccgg ataccaagtc 1081 catcgcacaa tgcagtttga agatggtgcc tcccttactg ttaactaccg ctacacctac 1141 gagggaagcc acatcaaagg agaggcccag gtgaagggga ctggtttccc tgctgacggt 1201 cctgtgatga ccaactcgct gaccgctgcg gactggtgca ggtcgaagaa gacttacccc 1261 aacgacaaaa ccatcatcag tacctttaag tggagttaca ccactggaaa tggcaagcgc 1321 taccggagca ctgcgcggac cacctacacc tttgccaagc caatggcggc taactatctg 1381 aagaaccagc cgatgtacgt gttccgtaag acggagctca agcactccaa gaccgagctc 1441 aacttcaagg agtggcaaaa ggcctttacc gatgtgatgg gcatggacga gctgtacaag 1501 gaattcatgg tgagcaaggg cgaggaggat aacatggcta tcatcaagga gttcatgcgc 1561 ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc 1621 gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc 1681 ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg 1741 aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg 1801 gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg 1861 caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc 1921 cccgtaatgc agaagaagac tatgggctgg gaggcctcct ccgagcggat gtaccccgag 1981 gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac 2041 gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac 2101 aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag 2161 tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta caagtaagct 2221 tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat cagaacgcag 2281 aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc cacctgaccc 2341 catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt ctccccatgc 2401 gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct 2461 ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccgggag 2521 cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc ccgccataaa 2581 ctgccaggca tcaaattaag cagaaggcca tcctgacgga tggccttttt gcgtttctac 2641 aaactctttt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa 2701 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt 2761 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg 2821 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg 2881 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg 2941 agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag 3001 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca 3061 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg 3121 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc 3181 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg 3241 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg 3301 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac 3361 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg 3421 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg 3481 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact 3541 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa 3601 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt 3661 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag 3721 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct 3781 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 3841 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg 3901 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct 3961 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc 4021 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg 4081 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 4141 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg 4201 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg 4261 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga 4321 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt 4381 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct 4441 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga 4501 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt 4561 ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc 4621 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg 4681 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg 4741 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac 4801 cgtcatcacc gaaacgcgcg aggcagcaga tcaattcgcg cgcgaaggcg aagcggcatg 4861 catttacgtt gacaccatcg aatggtgcaa aacctttcgc ggtatggcat gatagcgccc 4921 ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga 4981 gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc 5041 tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg 5101 cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct 5161 ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg 5221 tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt 5281 gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca 5341 ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc 5401 tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt 5461 ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc 5521 tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca 5581 gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca 5641 aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct 5701 gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt 5761 gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca 5821 ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca 5881 ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc 5941 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg 6001 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagcgcg 6061 aattgatctg gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca 6121 ggcagccatc ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt 6181 cgctcaaggc gcactcccgt tctggataat //