LOCUS       sequence-309818-        8057 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for LetB mutant F833Bpa.
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: pBEL1729
FEATURES             Location/Qualifiers
     source          1..8057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     541..560
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(580..657)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     863..882
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     966..985
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        966..984
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    985..1009
                     /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             3580..3597
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3663..3681)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3677..3724
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3761..4216
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3848..3867)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4058..4079
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(4291..4324)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4403..4507
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4508..4576,4577..5368)
                     /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(4726..4745)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5539..6127
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6028..6047
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6281..6298
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6313..6452
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6378..6397)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6538..6560
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6554..6745)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7554..8057,1..579))
                     /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 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
       61 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
      121 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
      181 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
      241 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
      301 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
      361 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
      421 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
      481 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
      541 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
      601 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
      661 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
      721 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
      781 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
      841 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
      901 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
      961 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat
     1021 aattttgttt aactttggcc caggcggcca ggagggtctc acatgcagga ccggggtaat
     1081 accgtcacca tcgactttat gtcggcggat ggtattgttc caggccgtac gcctgttcgt
     1141 tatcagggcg ttgaagtcgg aacagtgcag gatatcagcc tcagcgacga tcttcgtaag
     1201 attgaagtca aggtcagcat caagtccgat atgaaagatg cgctgcgcga agagactcag
     1261 ttctggctgg tgacgccaaa agcatcgttg gcaggtgtct ccgggctgga cgccctcgtc
     1321 ggtgggaact atatcggcat gatgccgggt aaaggtaaag agcaggatca ctttgtcgca
     1381 ctcgataccc aaccgaaata tcggctggac aatggcgatc tgatgatcca cctgcaagcc
     1441 cccgatctcg gttcgctgaa cagcggttca ttggtctatt tccgcaagat cccggtggga
     1501 aaagtctacg actatgccat caatcccaac aagcaaggcg tggtgattga tgtcctgatc
     1561 gagcggcgtt ttaccgatct ggtgaaaaaa ggtagccgtt tctggaacgt ttccggcgtt
     1621 gatgccaacg tcagtatcag tggcgcgaag gtgaaactgg aaagcctggc ggcactggtt
     1681 aacggtgcga ttgccttcga ttcaccagaa gagtcgaaac ctgccgaggc ggaagatacc
     1741 tttggtctgt atgaagatct ggcccacagc cagcgtggcg taataataaa actggaactg
     1801 ccgagtgggg ccggattaac cgccgactcg acgccgttaa tgtatcaggg gctggaagtc
     1861 ggacagctga ctaaactgga tttaaatcct ggtggtaaag tcacagggga aatgaccgtt
     1921 gatcccagcg tcgttaccct gcttcgtgaa aatacccgca tcgaattacg caacccgaaa
     1981 ttatccctta gcgatgctaa tctcagcgcc ctgctgaccg gcaaaacctt cgagctggtg
     2041 cccggcgatg gcgagccacg caaagagttc gttgttgtgc caggcgaaaa agcactgctg
     2101 catgaacctg atgttctgac gctgaccctg accgcgccgg aaagttacgg tattgatgcg
     2161 ggtcagccgc tcattcttca cggcgtgcag gtaggccagg ttatcgatcg taaactcacc
     2221 agcaaaggcg tcacctttac cgtcgccatc gagcctcagc atcgcgaact ggtaaaaggc
     2281 gatagcaaat ttgtcgtcaa cagccgtgtc gatgtgaagg tggggctgga tggcgttgag
     2341 tttctcggtg ccagcgcctc agaatggatc aatggcggga tacgtattct gccgggcgat
     2401 aaaggtgaga tgaaagccag ctatccactg tatgccaatt tggaaaaagc gctggagaac
     2461 agccttagcg atttacccac cacaaccgtg agtttgagtg cagagacgct gccggatgtg
     2521 caggcaggat cggtagtgct gtaccgtaaa tttgaagttg gtgaagtgat taccgtgcgt
     2581 ccgcgagcta acgcgtttga tatcgatctg catattaagc cggagtatcg caaccttctg
     2641 accagcaata gcgtgttctg ggcagaaggc ggggcgaaag ttcagctgaa tggtagtggt
     2701 ctgaccgtac aggcatcccc gctctccaga gcattaaagg gagccattag cttcgacaac
     2761 ctcagcggtg ccagcgccag tcagcgtaaa ggcgataagc gtattctgta tgcttccgaa
     2821 acagcggccc gtgcggttgg cgggcagatt acgcttcacg ctttcgatgc cggaaaactg
     2881 gcggtcggga tgccaattcg ctatctcggt attgatatcg ggcaaatcca gacgctggat
     2941 ctgattaccg cacgcaatga agtacaggca aaggcggtac tttacccgga atatgtccag
     3001 acctttgctc gcggtggtac gcgcttctca gtggtcacac cgcaaatttc ggcagctggc
     3061 gttgagcatc ttgatactat cctccagccg tatatcaacg tcgaaccagg ccggggcaat
     3121 cctcgccgcg actttgaatt acaagaggcc accattactg attcgcgtta cctggatggc
     3181 ttaagcatta ttgttgaagc gccggaagcc ggttcgttag gcatcggtac gcctgtgctg
     3241 ttccgtggtc tggaagtcgg tacggttaca ggaatgacgc tggggacatt gtcagatcgc
     3301 gtgatgattg cgatgcgcat cagtaaacgc tatcaacacc tggtgcgtaa caattccgtc
     3361 ttctggttgg catcgggtta cagtctggac tttggtctga cgggcggcgt agtgaaaacc
     3421 ggcaccttta accagtagat ccgtggcggc atcgccttcg ccacgcctcc gggtacgcca
     3481 ctggcaccga aagcccagga aggcaaacac ttcctgttgc aggaaagtga accgaaagag
     3541 tggcgtgaat ggggaactgc gcttcccaaa ggcgagaccc atcaccacca tcaccattaa
     3601 ggccaggccg gccgatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     3661 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     3721 tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca
     3781 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     3841 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     3901 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     3961 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     4021 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     4081 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     4141 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     4201 ttaacgctta caatttaggt ggcacttttc ggggaacgac gacctgcagc caagctagct
     4261 tggctggacg taaactcctc ttcagaccta ataacttcgt atagcataca ttatacgaag
     4321 ttatattaag ggttattgaa tatgatcgga attgggctgc aggaattcga tagcttggct
     4381 gcaggtcgac ctcgaggatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     4441 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     4501 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     4561 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     4621 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     4681 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     4741 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     4801 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     4861 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     4921 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     4981 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     5041 cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     5101 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     5161 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     5221 tggctctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     5281 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     5341 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     5401 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     5461 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     5521 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     5581 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     5641 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     5701 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     5761 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     5821 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     5881 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     5941 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     6001 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     6061 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     6121 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     6181 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     6241 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     6301 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     6361 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     6421 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     6481 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     6541 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     6601 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     6661 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     6721 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     6781 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     6841 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     6901 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     6961 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     7021 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     7081 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     7141 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     7201 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     7261 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     7321 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     7381 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     7441 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     7501 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     7561 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     7621 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     7681 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     7741 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     7801 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac
     7861 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     7921 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     7981 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     8041 gccagacgca gacgcgc
//
