LOCUS       sequence-309807-        8057 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for LetB mutant L358N.
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: pBEL1811
FEATURES             Location/Qualifiers
     source          1..8057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(88..1170)
                     /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"
     primer_bind     1132..1151
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1171..1248)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1454..1473
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1557..1576
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1557..1575
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1576..1600
                     /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             4171..4188
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4254..4272)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4268..4315
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4352..4807
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4439..4458)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4649..4670
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(4882..4915)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4994..5098
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5099..5167,5168..5959)
                     /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(5317..5336)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6130..6718
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6619..6638
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6872..6889
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6904..7043
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6969..6988)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7129..7151
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7145..7336)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
       61 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
      121 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
      181 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
      241 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
      301 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
      361 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
      421 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
      481 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
      541 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
      601 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
      661 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
      721 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
      781 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
      841 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
      901 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
      961 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     1021 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     1081 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     1141 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     1201 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     1261 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     1321 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     1381 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     1441 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     1501 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat
     1561 acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt
     1621 taactttggc ccaggcggcc aggagggtct cacatgcagg accggggtaa taccgtcacc
     1681 atcgacttta tgtcggcgga tggtattgtt ccaggccgta cgcctgttcg ttatcagggc
     1741 gttgaagtcg gaacagtgca ggatatcagc ctcagcgacg atcttcgtaa gattgaagtc
     1801 aaggtcagca tcaagtccga tatgaaagat gcgctgcgcg aagagactca gttctggctg
     1861 gtgacgccaa aagcatcgtt ggcaggtgtc tccgggctgg acgccctcgt cggtgggaac
     1921 tatatcggca tgatgccggg taaaggtaaa gagcaggatc actttgtcgc actcgatacc
     1981 caaccgaaat atcggctgga caatggcgat ctgatgatcc acctgcaagc ccccgatctc
     2041 ggttcgctga acagcggttc attggtctat ttccgcaaga tcccggtggg aaaagtctac
     2101 gactatgcca tcaatcccaa caagcaaggc gtggtgattg atgtcctgat cgagcggcgt
     2161 tttaccgatc tggtgaaaaa aggtagccgt ttctggaacg tttccggcgt tgatgccaac
     2221 gtcagtatca gtggcgcgaa ggtgaaactg gaaagcctgg cggcactggt taacggtgcg
     2281 attgccttcg attcaccaga agagtcgaaa cctgccgagg cggaagatac ctttggtctg
     2341 tatgaagatc tggcccacag ccagcgtggc gtaataataa aactggaact gccgagtggg
     2401 gccggattaa ccgccgactc gacgccgtta atgtatcagg ggctggaagt cggacagctg
     2461 actaaactgg atttaaatcc tggtggtaaa gtcacagggg aaatgaccgt tgatcccagc
     2521 gtcgttaccc tgcttcgtga aaatacccgc atcgaattac gcaacccgaa attatccctt
     2581 agcgatgcta atctcagcgc cctgaatacc ggcaaaacct tcgagctggt gcccggcgat
     2641 ggcgagccac gcaaagagtt cgttgttgtg ccaggcgaaa aagcactgct gcatgaacct
     2701 gatgttctga cgctgaccct gaccgcgccg gaaagttacg gtattgatgc gggtcagccg
     2761 ctcattcttc acggcgtgca ggtaggccag gttatcgatc gtaaactcac cagcaaaggc
     2821 gtcaccttta ccgtcgccat cgagcctcag catcgcgaac tggtaaaagg cgatagcaaa
     2881 tttgtcgtca acagccgtgt cgatgtgaag gtggggctgg atggcgttga gtttctcggt
     2941 gccagcgcct cagaatggat caatggcggg atacgtattc tgccgggcga taaaggtgag
     3001 atgaaagcca gctatccact gtatgccaat ttggaaaaag cgctggagaa cagccttagc
     3061 gatttaccca ccacaaccgt gagtttgagt gcagagacgc tgccggatgt gcaggcagga
     3121 tcggtagtgc tgtaccgtaa atttgaagtt ggtgaagtga ttaccgtgcg tccgcgagct
     3181 aacgcgtttg atatcgatct gcatattaag ccggagtatc gcaaccttct gaccagcaat
     3241 agcgtgttct gggcagaagg cggggcgaaa gttcagctga atggtagtgg tctgaccgta
     3301 caggcatccc cgctctccag agcattaaag ggagccatta gcttcgacaa cctcagcggt
     3361 gccagcgcca gtcagcgtaa aggcgataag cgtattctgt atgcttccga aacagcggcc
     3421 cgtgcggttg gcgggcagat tacgcttcac gctttcgatg ccggaaaact ggcggtcggg
     3481 atgccaattc gctatctcgg tattgatatc gggcaaatcc agacgctgga tctgattacc
     3541 gcacgcaatg aagtacaggc aaaggcggta ctttacccgg aatatgtcca gacctttgct
     3601 cgcggtggta cgcgcttctc agtggtcaca ccgcaaattt cggcagctgg cgttgagcat
     3661 cttgatacta tcctccagcc gtatatcaac gtcgaaccag gccggggcaa tcctcgccgc
     3721 gactttgaat tacaagaggc caccattact gattcgcgtt acctggatgg cttaagcatt
     3781 attgttgaag cgccggaagc cggttcgtta ggcatcggta cgcctgtgct gttccgtggt
     3841 ctggaagtcg gtacggttac aggaatgacg ctggggacat tgtcagatcg cgtgatgatt
     3901 gcgatgcgca tcagtaaacg ctatcaacac ctggtgcgta acaattccgt cttctggttg
     3961 gcatcgggtt acagtctgga ctttggtctg acgggcggcg tagtgaaaac cggcaccttt
     4021 aaccagttta tccgtggcgg catcgccttc gccacgcctc cgggtacgcc actggcaccg
     4081 aaagcccagg aaggcaaaca cttcctgttg caggaaagtg aaccgaaaga gtggcgtgaa
     4141 tggggaactg cgcttcccaa aggcgagacc catcaccacc atcaccatta aggccaggcc
     4201 ggccgatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
     4261 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     4321 aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     4381 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     4441 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     4501 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     4561 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     4621 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     4681 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     4741 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     4801 acaatttagg tggcactttt cggggaacga cgacctgcag ccaagctagc ttggctggac
     4861 gtaaactcct cttcagacct aataacttcg tatagcatac attatacgaa gttatattaa
     4921 gggttattga atatgatcgg aattgggctg caggaattcg atagcttggc tgcaggtcga
     4981 cctcgaggat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     5041 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     5101 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     5161 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     5221 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     5281 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     5341 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     5401 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     5461 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     5521 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     5581 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     5641 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     5701 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     5761 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtggctctcg
     5821 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     5881 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     5941 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     6001 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     6061 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     6121 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     6181 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     6241 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     6301 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6361 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6421 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6481 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     6541 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     6601 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     6661 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     6721 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     6781 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     6841 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     6901 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
     6961 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
     7021 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
     7081 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     7141 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     7201 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     7261 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
     7321 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
     7381 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
     7441 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
     7501 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
     7561 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
     7621 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
     7681 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
     7741 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
     7801 gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac
     7861 cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
     7921 tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca
     7981 cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
     8041 cccgcgccca ccggaag
//
