LOCUS       sequence-309811-        8057 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for LetB mutant W476pa.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8057)
  AUTHORS   Isom GL, Coudray N, MacRae MR, McManus CT, Ekiert DC, Bhabha G
  TITLE     LetB Structure Reveals a Tunnel for Lipid Transport across the 
            Bacterial Envelope.
  JOURNAL   Cell. 2020 Apr 30;181(3):653-664.e19. doi: 
            10.1016/j.cell.2020.03.030.
  PUBMED    32359438
REFERENCE   2  (bases 1 to 8057)
  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"; doi: 
            "10.1016/j.cell.2020.03"; journalName: "Cell"; date: "2020-04-30- 
            30"; volume: "181"; issue: "3"; pages: "653-664.e19"
COMMENT     Sequence Label: pBEL1726
FEATURES             Location/Qualifiers
     source          1..8057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     934..953
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(973..1050)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1256..1275
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1359..1378
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1359..1377
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1378..1402
                     /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)."
     CDS             3973..3990
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4056..4074)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4070..4117
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4154..4609
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4241..4260)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4451..4472
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(4684..4717)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4796..4900
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4901..4969,4970..5761)
                     /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     complement(5119..5138)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5932..6520
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6421..6440
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6674..6691
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6706..6845
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6771..6790)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6931..6953
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6947..7138)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7947..8057,1..972))
                     /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"
ORIGIN
        1 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
       61 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
      121 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc
      181 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
      241 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
      301 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
      361 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
      421 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
      481 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
      541 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
      601 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
      661 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
      721 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
      781 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
      841 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
      901 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
      961 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     1021 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
     1081 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     1141 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     1201 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     1261 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     1321 gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga
     1381 attgtgagcg gataacaatt cccctctaga aataattttg tttaactttg gcccaggcgg
     1441 ccaggagggt ctcacatgca ggaccggggt aataccgtca ccatcgactt tatgtcggcg
     1501 gatggtattg ttccaggccg tacgcctgtt cgttatcagg gcgttgaagt cggaacagtg
     1561 caggatatca gcctcagcga cgatcttcgt aagattgaag tcaaggtcag catcaagtcc
     1621 gatatgaaag atgcgctgcg cgaagagact cagttctggc tggtgacgcc aaaagcatcg
     1681 ttggcaggtg tctccgggct ggacgccctc gtcggtggga actatatcgg catgatgccg
     1741 ggtaaaggta aagagcagga tcactttgtc gcactcgata cccaaccgaa atatcggctg
     1801 gacaatggcg atctgatgat ccacctgcaa gcccccgatc tcggttcgct gaacagcggt
     1861 tcattggtct atttccgcaa gatcccggtg ggaaaagtct acgactatgc catcaatccc
     1921 aacaagcaag gcgtggtgat tgatgtcctg atcgagcggc gttttaccga tctggtgaaa
     1981 aaaggtagcc gtttctggaa cgtttccggc gttgatgcca acgtcagtat cagtggcgcg
     2041 aaggtgaaac tggaaagcct ggcggcactg gttaacggtg cgattgcctt cgattcacca
     2101 gaagagtcga aacctgccga ggcggaagat acctttggtc tgtatgaaga tctggcccac
     2161 agccagcgtg gcgtaataat aaaactggaa ctgccgagtg gggccggatt aaccgccgac
     2221 tcgacgccgt taatgtatca ggggctggaa gtcggacagc tgactaaact ggatttaaat
     2281 cctggtggta aagtcacagg ggaaatgacc gttgatccca gcgtcgttac cctgcttcgt
     2341 gaaaataccc gcatcgaatt acgcaacccg aaattatccc ttagcgatgc taatctcagc
     2401 gccctgctga ccggcaaaac cttcgagctg gtgcccggcg atggcgagcc acgcaaagag
     2461 ttcgttgttg tgccaggcga aaaagcactg ctgcatgaac ctgatgttct gacgctgacc
     2521 ctgaccgcgc cggaaagtta cggtattgat gcgggtcagc cgctcattct tcacggcgtg
     2581 caggtaggcc aggttatcga tcgtaaactc accagcaaag gcgtcacctt taccgtcgcc
     2641 atcgagcctc agcatcgcga actggtaaaa ggcgatagca aatttgtcgt caacagccgt
     2701 gtcgatgtga aggtggggct ggatggcgtt gagtttctcg gtgccagcgc ctcagaatag
     2761 atcaatggcg ggatacgtat tctgccgggc gataaaggtg agatgaaagc cagctatcca
     2821 ctgtatgcca atttggaaaa agcgctggag aacagcctta gcgatttacc caccacaacc
     2881 gtgagtttga gtgcagagac gctgccggat gtgcaggcag gatcggtagt gctgtaccgt
     2941 aaatttgaag ttggtgaagt gattaccgtg cgtccgcgag ctaacgcgtt tgatatcgat
     3001 ctgcatatta agccggagta tcgcaacctt ctgaccagca atagcgtgtt ctgggcagaa
     3061 ggcggggcga aagttcagct gaatggtagt ggtctgaccg tacaggcatc cccgctctcc
     3121 agagcattaa agggagccat tagcttcgac aacctcagcg gtgccagcgc cagtcagcgt
     3181 aaaggcgata agcgtattct gtatgcttcc gaaacagcgg cccgtgcggt tggcgggcag
     3241 attacgcttc acgctttcga tgccggaaaa ctggcggtcg ggatgccaat tcgctatctc
     3301 ggtattgata tcgggcaaat ccagacgctg gatctgatta ccgcacgcaa tgaagtacag
     3361 gcaaaggcgg tactttaccc ggaatatgtc cagacctttg ctcgcggtgg tacgcgcttc
     3421 tcagtggtca caccgcaaat ttcggcagct ggcgttgagc atcttgatac tatcctccag
     3481 ccgtatatca acgtcgaacc aggccggggc aatcctcgcc gcgactttga attacaagag
     3541 gccaccatta ctgattcgcg ttacctggat ggcttaagca ttattgttga agcgccggaa
     3601 gccggttcgt taggcatcgg tacgcctgtg ctgttccgtg gtctggaagt cggtacggtt
     3661 acaggaatga cgctggggac attgtcagat cgcgtgatga ttgcgatgcg catcagtaaa
     3721 cgctatcaac acctggtgcg taacaattcc gtcttctggt tggcatcggg ttacagtctg
     3781 gactttggtc tgacgggcgg cgtagtgaaa accggcacct ttaaccagtt tatccgtggc
     3841 ggcatcgcct tcgccacgcc tccgggtacg ccactggcac cgaaagccca ggaaggcaaa
     3901 cacttcctgt tgcaggaaag tgaaccgaaa gagtggcgtg aatggggaac tgcgcttccc
     3961 aaaggcgaga cccatcacca ccatcaccat taaggccagg ccggccgatc cggctgctaa
     4021 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     4081 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     4141 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     4201 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     4261 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     4321 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     4381 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     4441 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     4501 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     4561 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     4621 ttcggggaac gacgacctgc agccaagcta gcttggctgg acgtaaactc ctcttcagac
     4681 ctaataactt cgtatagcat acattatacg aagttatatt aagggttatt gaatatgatc
     4741 ggaattgggc tgcaggaatt cgatagcttg gctgcaggtc gacctcgagg atgtgcgcgg
     4801 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     4861 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     4921 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     4981 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     5041 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     5101 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     5161 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     5221 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     5281 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     5341 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     5401 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac
     5461 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     5521 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     5581 gtttattgct gataaatctg gagccggtga gcgtggctct cgcggtatca ttgcagcact
     5641 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     5701 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     5761 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     5821 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     5881 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     5941 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     6001 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     6061 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     6121 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     6181 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     6241 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     6301 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     6361 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     6421 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     6481 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     6541 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     6601 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     6661 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     6721 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
     6781 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     6841 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     6901 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     6961 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     7021 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     7081 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     7141 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     7201 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     7261 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     7321 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     7381 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     7441 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     7501 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     7561 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg
     7621 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     7681 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     7741 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     7801 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     7861 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     7921 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     7981 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     8041 agggtggttt ttctttt
//
