LOCUS       sequence-422157-        6399 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  OmpT_native membrane expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6399)
  AUTHORS   Rath P, Hermann A, Schaefer R, Agustoni E, Vonach JM, Siegrist M, 
            Miscenic C, Tschumi A, Roth D, Bieniossek C, Hiller S
  TITLE     High-throughput screening of BAM inhibitors in native membrane 
            environment.
  JOURNAL   Nat Commun. 2023 Sep 13;14(1):5648. doi: 10.1038/s41467-023-41445-w.
  PUBMED    37704632
REFERENCE   2  (bases 1 to 6399)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2023-09-13"; volume: "14(1):5648. doi"; pages: " 
            10.1038/s41467-023-41445-w"
COMMENT     Sequence Label: OmpT
FEATURES             Location/Qualifiers
     source          1..6399
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(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(1077..1097)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(1107..1124)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1207..1229
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1244..1268
                     /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(1268..1287)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1269..1287)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1371..1390)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1596..1673
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1674..2756
                     /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(1693..1712)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3565..3756
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3750..3772)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3858..3997
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3913..3932
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4012..4029)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4183..4771)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4263..4282)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4942..5733,5734..5802))
                     /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     5565..5584
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5803..5907)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5933..6388)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6070..6091)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6282..6301
                     /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 taaaatgtgt acttaagacc agcagtagtg atgaagttat agttttctat acctgctcca
      241 tttttgctgt agtctgaagt gttattattg tgatcataaa gtgaagtatt acctttttta
      301 ttcgtaaccc gattccatgc gccttcaaca taaacttttg cgttaggtgt gacgtaataa
      361 cctgcattga ctgcaacaga atagtaattt tggtctttga ccttactgcg ataagtgatt
      421 ctttttcccg ggtcatagtg ttcatcgtta tcagatgatt ccacccagcc gctgtattta
      481 aatgtgccac cgagttcaaa atcttcataa cgataacttc cagtcaagcc aatgtagggc
      541 attttaaaac gttgtttgta gccgattgct ctttctccat tcgggaagga gccgatatca
      601 tctctgaatc cctcctcaga actgtagata taggaaccac ctctggctgt aaagctataa
      661 cggctttcct gatatccggc catgagtccc aggcggtaat tgggttcgtt gaggagccag
      721 cctttgatat tcagatcaaa ttcgttggca taattgagtt gtgtatcagg gtgtctactt
      781 tcatccgtcc aggttccggg gttactggaa tccatccagt cctgatcgac catattgcca
      841 cctcggctgc cgagagttgt ccagccagca gccccgatag atatctgggg catcaaatcc
      901 caattaattg cacctttaat aattgcagcg ttattgaatt tccagtcgag ttgactgact
      961 tttcggcctc cttcttcggc tagataaaca cgctcttttg tttttccgct cagagttcca
     1021 agactaatgt ccgcatttat gttgtcagga gtaaacgata aagtctcggt agaggatccc
     1081 tggaagtaca ggttttcgcc gctgctgtga tgatgatgat gatggctgct gcccatggcc
     1141 tgtgcgcagg ccattcccgc acacatcacc agtgcggtac agggtaataa ctttttcata
     1201 tgtatatctc cttcttaaag ttaaacaaaa ttatttctag aggggaattg ttatccgctc
     1261 acaattcccc tatagtgagt cgtattaatt tcgcgggatc gagatctcga tcctctacgc
     1321 cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc
     1381 cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg
     1441 cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg ggcgccatct ccttgcatgc
     1501 accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat
     1561 gcaggagtcg cataagggag agcgtcgaga tcccggacac catcgaatgg cgcaaaacct
     1621 ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac
     1681 cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg
     1741 tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg
     1801 cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc
     1861 tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga
     1921 ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg
     1981 gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga
     2041 tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg
     2101 ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt attttctccc
     2161 atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg
     2221 cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata
     2281 aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca
     2341 tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc
     2401 tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc
     2461 gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata
     2521 tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc
     2581 gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac
     2641 tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg
     2701 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     2761 aacgcaatta atgtaagtta gctcactcat taggcaccgg gatctcgacc gatgcccttg
     2821 agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca
     2881 cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc gctctgggtc
     2941 attttcggcg aggaccgctt tcgctggagc gcgacgatga tcggcctgtc gcttgcggta
     3001 ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc
     3061 ggcgagaagc aggccattat cgccggcatg gcggccccac gggtgcgcat gatcgtgctc
     3121 ctgtcgttga ggacccggct aggctggcgg ggttgcctta ctggttagca gaatgaatca
     3181 ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca
     3241 acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc
     3301 tgcaccatta tgttccggat ctgcatcgca ggatgctgct ggctaccctg tggaacacct
     3361 acatctgtat taacgaagcg ctggcattga ccctgagtga tttttctctg gtcccgccgc
     3421 atccataccg ccagttgttt accctcacaa cgttccagta accgggcatg ttcatcatca
     3481 gtaacccgta tcgtgagcat cctctctcgt ttcatcggta tcattacccc catgaacaga
     3541 aatccccctt acacggaggc atcagtgacc aaacaggaaa aaaccgccct taacatggcc
     3601 cgctttatca gaagccagac attaacgctt ctggagaaac tcaacgagct ggacgcggat
     3661 gaacaggcag acatctgtga atcgcttcac gaccacgctg atgagcttta ccgcagctgc
     3721 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     3781 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     3841 gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact
     3901 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccattgc ggtgtgaaat
     3961 accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac
     4021 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4081 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4141 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4201 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4261 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4321 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4381 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4441 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4501 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4561 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     4621 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4681 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4741 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     4801 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     4861 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     4921 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     4981 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5041 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5101 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5161 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5221 gccagttaat agtttgcgca acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc
     5281 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5341 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5401 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     5461 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     5521 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     5581 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     5641 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     5701 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     5761 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     5821 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     5881 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta
     5941 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     6001 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     6061 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     6121 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     6181 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     6241 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     6301 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     6361 gcgcttaatg cgccgctaca gggcgcgtcc cattcgcca
//
