LOCUS       sequence-309822-        8057 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for LetB mutant E854Bpa.
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: pBEL1731
FEATURES             Location/Qualifiers
     source          1..8057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     932..951
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(971..1048)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1254..1273
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1357..1376
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1357..1375
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1376..1400
                     /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             3971..3988
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4054..4072)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4068..4115
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4152..4607
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4239..4258)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4449..4470
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(4682..4715)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4794..4898
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4899..4967,4968..5759)
                     /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(5117..5136)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5930..6518
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6419..6438
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6672..6689
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6704..6843
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6769..6788)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6929..6951
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6945..7136)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7945..8057,1..970))
                     /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 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
       61 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
      121 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
      181 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
      241 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
      301 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
      361 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
      421 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
      481 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
      541 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
      601 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
      661 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
      721 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
      781 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
      841 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
      901 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
      961 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     1021 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     1081 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     1141 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     1201 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     1261 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     1321 ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat
     1381 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttggc ccaggcggcc
     1441 aggagggtct cacatgcagg accggggtaa taccgtcacc atcgacttta tgtcggcgga
     1501 tggtattgtt ccaggccgta cgcctgttcg ttatcagggc gttgaagtcg gaacagtgca
     1561 ggatatcagc ctcagcgacg atcttcgtaa gattgaagtc aaggtcagca tcaagtccga
     1621 tatgaaagat gcgctgcgcg aagagactca gttctggctg gtgacgccaa aagcatcgtt
     1681 ggcaggtgtc tccgggctgg acgccctcgt cggtgggaac tatatcggca tgatgccggg
     1741 taaaggtaaa gagcaggatc actttgtcgc actcgatacc caaccgaaat atcggctgga
     1801 caatggcgat ctgatgatcc acctgcaagc ccccgatctc ggttcgctga acagcggttc
     1861 attggtctat ttccgcaaga tcccggtggg aaaagtctac gactatgcca tcaatcccaa
     1921 caagcaaggc gtggtgattg atgtcctgat cgagcggcgt tttaccgatc tggtgaaaaa
     1981 aggtagccgt ttctggaacg tttccggcgt tgatgccaac gtcagtatca gtggcgcgaa
     2041 ggtgaaactg gaaagcctgg cggcactggt taacggtgcg attgccttcg attcaccaga
     2101 agagtcgaaa cctgccgagg cggaagatac ctttggtctg tatgaagatc tggcccacag
     2161 ccagcgtggc gtaataataa aactggaact gccgagtggg gccggattaa ccgccgactc
     2221 gacgccgtta atgtatcagg ggctggaagt cggacagctg actaaactgg atttaaatcc
     2281 tggtggtaaa gtcacagggg aaatgaccgt tgatcccagc gtcgttaccc tgcttcgtga
     2341 aaatacccgc atcgaattac gcaacccgaa attatccctt agcgatgcta atctcagcgc
     2401 cctgctgacc ggcaaaacct tcgagctggt gcccggcgat ggcgagccac gcaaagagtt
     2461 cgttgttgtg ccaggcgaaa aagcactgct gcatgaacct gatgttctga cgctgaccct
     2521 gaccgcgccg gaaagttacg gtattgatgc gggtcagccg ctcattcttc acggcgtgca
     2581 ggtaggccag gttatcgatc gtaaactcac cagcaaaggc gtcaccttta ccgtcgccat
     2641 cgagcctcag catcgcgaac tggtaaaagg cgatagcaaa tttgtcgtca acagccgtgt
     2701 cgatgtgaag gtggggctgg atggcgttga gtttctcggt gccagcgcct cagaatggat
     2761 caatggcggg atacgtattc tgccgggcga taaaggtgag atgaaagcca gctatccact
     2821 gtatgccaat ttggaaaaag cgctggagaa cagccttagc gatttaccca ccacaaccgt
     2881 gagtttgagt gcagagacgc tgccggatgt gcaggcagga tcggtagtgc tgtaccgtaa
     2941 atttgaagtt ggtgaagtga ttaccgtgcg tccgcgagct aacgcgtttg atatcgatct
     3001 gcatattaag ccggagtatc gcaaccttct gaccagcaat agcgtgttct gggcagaagg
     3061 cggggcgaaa gttcagctga atggtagtgg tctgaccgta caggcatccc cgctctccag
     3121 agcattaaag ggagccatta gcttcgacaa cctcagcggt gccagcgcca gtcagcgtaa
     3181 aggcgataag cgtattctgt atgcttccga aacagcggcc cgtgcggttg gcgggcagat
     3241 tacgcttcac gctttcgatg ccggaaaact ggcggtcggg atgccaattc gctatctcgg
     3301 tattgatatc gggcaaatcc agacgctgga tctgattacc gcacgcaatg aagtacaggc
     3361 aaaggcggta ctttacccgg aatatgtcca gacctttgct cgcggtggta cgcgcttctc
     3421 agtggtcaca ccgcaaattt cggcagctgg cgttgagcat cttgatacta tcctccagcc
     3481 gtatatcaac gtcgaaccag gccggggcaa tcctcgccgc gactttgaat tacaagaggc
     3541 caccattact gattcgcgtt acctggatgg cttaagcatt attgttgaag cgccggaagc
     3601 cggttcgtta ggcatcggta cgcctgtgct gttccgtggt ctggaagtcg gtacggttac
     3661 aggaatgacg ctggggacat tgtcagatcg cgtgatgatt gcgatgcgca tcagtaaacg
     3721 ctatcaacac ctggtgcgta acaattccgt cttctggttg gcatcgggtt acagtctgga
     3781 ctttggtctg acgggcggcg tagtgaaaac cggcaccttt aaccagttta tccgtggcgg
     3841 catcgccttc gccacgcctc cgggtacgcc actggcaccg aaagcccagt agggcaaaca
     3901 cttcctgttg caggaaagtg aaccgaaaga gtggcgtgaa tggggaactg cgcttcccaa
     3961 aggcgagacc catcaccacc atcaccatta aggccaggcc ggccgatccg gctgctaaca
     4021 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     4081 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     4141 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     4201 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     4261 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     4321 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     4381 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     4441 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     4501 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     4561 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     4621 cggggaacga cgacctgcag ccaagctagc ttggctggac gtaaactcct cttcagacct
     4681 aataacttcg tatagcatac attatacgaa gttatattaa gggttattga atatgatcgg
     4741 aattgggctg caggaattcg atagcttggc tgcaggtcga cctcgaggat gtgcgcggaa
     4801 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     4861 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     4921 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     4981 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     5041 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     5101 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     5161 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     5221 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     5281 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     5341 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     5401 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt
     5461 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     5521 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     5581 ttattgctga taaatctgga gccggtgagc gtggctctcg cggtatcatt gcagcactgg
     5641 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     5701 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     5761 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     5821 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     5881 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     5941 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     6001 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     6061 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     6121 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     6181 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     6241 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     6301 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     6361 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     6421 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     6481 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     6541 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     6601 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     6661 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     6721 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     6781 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     6841 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     6901 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     6961 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     7021 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     7081 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     7141 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     7201 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     7261 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     7321 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     7381 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     7441 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     7501 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     7561 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg
     7621 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     7681 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
     7741 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     7801 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     7861 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     7921 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     7981 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     8041 ggtggttttt cttttca
//
