LOCUS       sequence-309817-        8057 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for LetB mutant F818Bpa.
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: pBEL1728
FEATURES             Location/Qualifiers
     source          1..8057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     975..994
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1014..1091)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1297..1316
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1400..1419
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1400..1418
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1419..1443
                     /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             4014..4031
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4097..4115)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4111..4158
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4195..4650
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4282..4301)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4492..4513
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(4725..4758)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4837..4941
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4942..5010,5011..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     complement(5160..5179)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5973..6561
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6462..6481
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6715..6732
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6747..6886
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6812..6831)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6972..6994
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6988..7179)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7988..8057,1..1013))
                     /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 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
       61 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
      121 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
      181 tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg
      241 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
      301 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
      361 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
      421 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
      481 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
      541 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
      601 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
      661 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
      721 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
      781 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
      841 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
      901 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
      961 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     1021 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     1081 tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc
     1141 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat
     1201 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct
     1261 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc
     1321 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga
     1381 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
     1441 tcccctctag aaataatttt gtttaacttt ggcccaggcg gccaggaggg tctcacatgc
     1501 aggaccgggg taataccgtc accatcgact ttatgtcggc ggatggtatt gttccaggcc
     1561 gtacgcctgt tcgttatcag ggcgttgaag tcggaacagt gcaggatatc agcctcagcg
     1621 acgatcttcg taagattgaa gtcaaggtca gcatcaagtc cgatatgaaa gatgcgctgc
     1681 gcgaagagac tcagttctgg ctggtgacgc caaaagcatc gttggcaggt gtctccgggc
     1741 tggacgccct cgtcggtggg aactatatcg gcatgatgcc gggtaaaggt aaagagcagg
     1801 atcactttgt cgcactcgat acccaaccga aatatcggct ggacaatggc gatctgatga
     1861 tccacctgca agcccccgat ctcggttcgc tgaacagcgg ttcattggtc tatttccgca
     1921 agatcccggt gggaaaagtc tacgactatg ccatcaatcc caacaagcaa ggcgtggtga
     1981 ttgatgtcct gatcgagcgg cgttttaccg atctggtgaa aaaaggtagc cgtttctgga
     2041 acgtttccgg cgttgatgcc aacgtcagta tcagtggcgc gaaggtgaaa ctggaaagcc
     2101 tggcggcact ggttaacggt gcgattgcct tcgattcacc agaagagtcg aaacctgccg
     2161 aggcggaaga tacctttggt ctgtatgaag atctggccca cagccagcgt ggcgtaataa
     2221 taaaactgga actgccgagt ggggccggat taaccgccga ctcgacgccg ttaatgtatc
     2281 aggggctgga agtcggacag ctgactaaac tggatttaaa tcctggtggt aaagtcacag
     2341 gggaaatgac cgttgatccc agcgtcgtta ccctgcttcg tgaaaatacc cgcatcgaat
     2401 tacgcaaccc gaaattatcc cttagcgatg ctaatctcag cgccctgctg accggcaaaa
     2461 ccttcgagct ggtgcccggc gatggcgagc cacgcaaaga gttcgttgtt gtgccaggcg
     2521 aaaaagcact gctgcatgaa cctgatgttc tgacgctgac cctgaccgcg ccggaaagtt
     2581 acggtattga tgcgggtcag ccgctcattc ttcacggcgt gcaggtaggc caggttatcg
     2641 atcgtaaact caccagcaaa ggcgtcacct ttaccgtcgc catcgagcct cagcatcgcg
     2701 aactggtaaa aggcgatagc aaatttgtcg tcaacagccg tgtcgatgtg aaggtggggc
     2761 tggatggcgt tgagtttctc ggtgccagcg cctcagaatg gatcaatggc gggatacgta
     2821 ttctgccggg cgataaaggt gagatgaaag ccagctatcc actgtatgcc aatttggaaa
     2881 aagcgctgga gaacagcctt agcgatttac ccaccacaac cgtgagtttg agtgcagaga
     2941 cgctgccgga tgtgcaggca ggatcggtag tgctgtaccg taaatttgaa gttggtgaag
     3001 tgattaccgt gcgtccgcga gctaacgcgt ttgatatcga tctgcatatt aagccggagt
     3061 atcgcaacct tctgaccagc aatagcgtgt tctgggcaga aggcggggcg aaagttcagc
     3121 tgaatggtag tggtctgacc gtacaggcat ccccgctctc cagagcatta aagggagcca
     3181 ttagcttcga caacctcagc ggtgccagcg ccagtcagcg taaaggcgat aagcgtattc
     3241 tgtatgcttc cgaaacagcg gcccgtgcgg ttggcgggca gattacgctt cacgctttcg
     3301 atgccggaaa actggcggtc gggatgccaa ttcgctatct cggtattgat atcgggcaaa
     3361 tccagacgct ggatctgatt accgcacgca atgaagtaca ggcaaaggcg gtactttacc
     3421 cggaatatgt ccagaccttt gctcgcggtg gtacgcgctt ctcagtggtc acaccgcaaa
     3481 tttcggcagc tggcgttgag catcttgata ctatcctcca gccgtatatc aacgtcgaac
     3541 caggccgggg caatcctcgc cgcgactttg aattacaaga ggccaccatt actgattcgc
     3601 gttacctgga tggcttaagc attattgttg aagcgccgga agccggttcg ttaggcatcg
     3661 gtacgcctgt gctgttccgt ggtctggaag tcggtacggt tacaggaatg acgctgggga
     3721 cattgtcaga tcgcgtgatg attgcgatgc gcatcagtaa acgctatcaa cacctggtgc
     3781 gtaacaattc cgtcttctgg ttggcatcgg gttacagtct ggactagggt ctgacgggcg
     3841 gcgtagtgaa aaccggcacc tttaaccagt ttatccgtgg cggcatcgcc ttcgccacgc
     3901 ctccgggtac gccactggca ccgaaagccc aggaaggcaa acacttcctg ttgcaggaaa
     3961 gtgaaccgaa agagtggcgt gaatggggaa ctgcgcttcc caaaggcgag acccatcacc
     4021 accatcacca ttaaggccag gccggccgat ccggctgcta acaaagcccg aaaggaagct
     4081 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     4141 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
     4201 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     4261 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     4321 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     4381 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     4441 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     4501 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     4561 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     4621 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa cgacgacctg
     4681 cagccaagct agcttggctg gacgtaaact cctcttcaga cctaataact tcgtatagca
     4741 tacattatac gaagttatat taagggttat tgaatatgat cggaattggg ctgcaggaat
     4801 tcgatagctt ggctgcaggt cgacctcgag gatgtgcgcg gaacccctat ttgtttattt
     4861 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     4921 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     4981 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     5041 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     5101 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     5161 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     5221 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     5281 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     5341 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     5401 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     5461 gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact
     5521 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     5581 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     5641 ggagccggtg agcgtggctc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     5701 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     5761 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     5821 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     5881 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     5941 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     6001 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     6061 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     6121 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     6181 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     6241 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     6301 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     6361 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     6421 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     6481 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     6541 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     6601 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     6661 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     6721 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     6781 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     6841 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     6901 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     6961 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     7021 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     7081 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     7141 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     7201 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     7261 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
     7321 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
     7381 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     7441 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
     7501 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
     7561 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
     7621 caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc
     7681 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
     7741 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
     7801 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat
     7861 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     7921 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     7981 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     8041 cggccaacgc gcgggga
//
