LOCUS       sequence-309771-        8057 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for LetB mutant V247N.
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: pBEL1808
FEATURES             Location/Qualifiers
     source          1..8057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     563..582
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(602..679)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     885..904
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     988..1007
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        988..1006
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1007..1031
                     /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             3602..3619
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3685..3703)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3699..3746
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3783..4238
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3870..3889)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4080..4101
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(4313..4346)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4425..4529
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4530..4598,4599..5390)
                     /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(4748..4767)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5561..6149
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6050..6069
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6303..6320
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6335..6474
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6400..6419)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6560..6582
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6576..6767)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7576..8057,1..601))
                     /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 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
       61 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
      121 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
      181 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
      241 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
      301 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
      361 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
      421 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
      481 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
      541 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
      601 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
      661 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
      721 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
      781 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
      841 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
      901 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
      961 aggatcgaga tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg
     1021 ataacaattc ccctctagaa ataattttgt ttaactttgg cccaggcggc caggagggtc
     1081 tcacatgcag gaccggggta ataccgtcac catcgacttt atgtcggcgg atggtattgt
     1141 tccaggccgt acgcctgttc gttatcaggg cgttgaagtc ggaacagtgc aggatatcag
     1201 cctcagcgac gatcttcgta agattgaagt caaggtcagc atcaagtccg atatgaaaga
     1261 tgcgctgcgc gaagagactc agttctggct ggtgacgcca aaagcatcgt tggcaggtgt
     1321 ctccgggctg gacgccctcg tcggtgggaa ctatatcggc atgatgccgg gtaaaggtaa
     1381 agagcaggat cactttgtcg cactcgatac ccaaccgaaa tatcggctgg acaatggcga
     1441 tctgatgatc cacctgcaag cccccgatct cggttcgctg aacagcggtt cattggtcta
     1501 tttccgcaag atcccggtgg gaaaagtcta cgactatgcc atcaatccca acaagcaagg
     1561 cgtggtgatt gatgtcctga tcgagcggcg ttttaccgat ctggtgaaaa aaggtagccg
     1621 tttctggaac gtttccggcg ttgatgccaa cgtcagtatc agtggcgcga aggtgaaact
     1681 ggaaagcctg gcggcactga ataacggtgc gattgccttc gattcaccag aagagtcgaa
     1741 acctgccgag gcggaagata cctttggtct gtatgaagat ctggcccaca gccagcgtgg
     1801 cgtaataata aaactggaac tgccgagtgg ggccggatta accgccgact cgacgccgtt
     1861 aatgtatcag gggctggaag tcggacagct gactaaactg gatttaaatc ctggtggtaa
     1921 agtcacaggg gaaatgaccg ttgatcccag cgtcgttacc ctgcttcgtg aaaatacccg
     1981 catcgaatta cgcaacccga aattatccct tagcgatgct aatctcagcg ccctgctgac
     2041 cggcaaaacc ttcgagctgg tgcccggcga tggcgagcca cgcaaagagt tcgttgttgt
     2101 gccaggcgaa aaagcactgc tgcatgaacc tgatgttctg acgctgaccc tgaccgcgcc
     2161 ggaaagttac ggtattgatg cgggtcagcc gctcattctt cacggcgtgc aggtaggcca
     2221 ggttatcgat cgtaaactca ccagcaaagg cgtcaccttt accgtcgcca tcgagcctca
     2281 gcatcgcgaa ctggtaaaag gcgatagcaa atttgtcgtc aacagccgtg tcgatgtgaa
     2341 ggtggggctg gatggcgttg agtttctcgg tgccagcgcc tcagaatgga tcaatggcgg
     2401 gatacgtatt ctgccgggcg ataaaggtga gatgaaagcc agctatccac tgtatgccaa
     2461 tttggaaaaa gcgctggaga acagccttag cgatttaccc accacaaccg tgagtttgag
     2521 tgcagagacg ctgccggatg tgcaggcagg atcggtagtg ctgtaccgta aatttgaagt
     2581 tggtgaagtg attaccgtgc gtccgcgagc taacgcgttt gatatcgatc tgcatattaa
     2641 gccggagtat cgcaaccttc tgaccagcaa tagcgtgttc tgggcagaag gcggggcgaa
     2701 agttcagctg aatggtagtg gtctgaccgt acaggcatcc ccgctctcca gagcattaaa
     2761 gggagccatt agcttcgaca acctcagcgg tgccagcgcc agtcagcgta aaggcgataa
     2821 gcgtattctg tatgcttccg aaacagcggc ccgtgcggtt ggcgggcaga ttacgcttca
     2881 cgctttcgat gccggaaaac tggcggtcgg gatgccaatt cgctatctcg gtattgatat
     2941 cgggcaaatc cagacgctgg atctgattac cgcacgcaat gaagtacagg caaaggcggt
     3001 actttacccg gaatatgtcc agacctttgc tcgcggtggt acgcgcttct cagtggtcac
     3061 accgcaaatt tcggcagctg gcgttgagca tcttgatact atcctccagc cgtatatcaa
     3121 cgtcgaacca ggccggggca atcctcgccg cgactttgaa ttacaagagg ccaccattac
     3181 tgattcgcgt tacctggatg gcttaagcat tattgttgaa gcgccggaag ccggttcgtt
     3241 aggcatcggt acgcctgtgc tgttccgtgg tctggaagtc ggtacggtta caggaatgac
     3301 gctggggaca ttgtcagatc gcgtgatgat tgcgatgcgc atcagtaaac gctatcaaca
     3361 cctggtgcgt aacaattccg tcttctggtt ggcatcgggt tacagtctgg actttggtct
     3421 gacgggcggc gtagtgaaaa ccggcacctt taaccagttt atccgtggcg gcatcgcctt
     3481 cgccacgcct ccgggtacgc cactggcacc gaaagcccag gaaggcaaac acttcctgtt
     3541 gcaggaaagt gaaccgaaag agtggcgtga atggggaact gcgcttccca aaggcgagac
     3601 ccatcaccac catcaccatt aaggccaggc cggccgatcc ggctgctaac aaagcccgaa
     3661 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     3721 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
     3781 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     3841 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     3901 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     3961 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     4021 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     4081 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     4141 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     4201 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaacg
     4261 acgacctgca gccaagctag cttggctgga cgtaaactcc tcttcagacc taataacttc
     4321 gtatagcata cattatacga agttatatta agggttattg aatatgatcg gaattgggct
     4381 gcaggaattc gatagcttgg ctgcaggtcg acctcgagga tgtgcgcgga acccctattt
     4441 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     4501 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     4561 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     4621 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     4681 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     4741 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     4801 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     4861 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     4921 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     4981 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     5041 taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac
     5101 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     5161 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     5221 ataaatctgg agccggtgag cgtggctctc gcggtatcat tgcagcactg gggccagatg
     5281 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     5341 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     5401 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     5461 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     5521 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     5581 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     5641 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     5701 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     5761 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     5821 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     5881 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     5941 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     6001 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     6061 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     6121 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     6181 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     6241 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     6301 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     6361 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     6421 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     6481 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     6541 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     6601 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     6661 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     6721 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     6781 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     6841 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     6901 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     6961 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     7021 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     7081 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     7141 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     7201 ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc
     7261 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
     7321 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc
     7381 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag
     7441 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
     7501 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     7561 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     7621 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     7681 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     7741 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     7801 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
     7861 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     7921 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     7981 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     8041 agtgagatat ttatgcc
//
