LOCUS       sequence-309711-        6995 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for LetAB with 4 ring (rings 1567) LetB (without 
            TM region) mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6995)
  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 6995)
  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: pBEL2002
FEATURES             Location/Qualifiers
     source          1..6995
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     973..992
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1012..1089)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1295..1314
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1398..1417
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1398..1416
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1417..1441
                     /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             2950..2967
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3033..3051)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3047..3094
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3131..3586
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3218..3237)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3428..3449
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(3661..3694)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3773..3877
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3878..3946,3947..4738)
                     /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(4096..4115)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4909..5497
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5398..5417
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5651..5668
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5683..5822
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5748..5767)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5908..5930
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5924..6115)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6924..6995,1..1011))
                     /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 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
       61 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
      121 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
      181 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
      241 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
      301 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
      361 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
      421 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
      481 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
      541 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
      601 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
      661 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
      721 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
      781 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
      841 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
      901 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
      961 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     1021 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     1081 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     1141 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     1201 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     1261 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     1321 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
     1381 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
     1441 ccctctagaa ataattttgt ttaactttgg cccaggcggc caggagggtc tcacatgcag
     1501 gaccggggta ataccgtcac catcgacttt atgtcggcgg atggtattgt tccaggccgt
     1561 acgcctgttc gttatcaggg cgttgaagtc ggaacagtgc aggatatcag cctcagcgac
     1621 gatcttcgta agattgaagt caaggtcagc atcaagtccg atatgaaaga tgcgctgcgc
     1681 gaagagactc agttctggct ggtgacgcca aaagcatcgt tggcaggtgt ctccgggctg
     1741 gacgccctcg tcggtgggaa ctatatcggc atgatgccgg gtaaaggtaa agagcaggat
     1801 cactttgtcg cactcgatac ccaaccgaaa tatcggctgg acaatggcga tacaaccgtg
     1861 agtttgagtg cagagacgct gccggatgtg caggcaggat cggtagtgct gtaccgtaaa
     1921 tttgaagttg gtgaagtgat taccgtgcgt ccgcgagcta acgcgtttga tatcgatctg
     1981 catattaagc cggagtatcg caaccttctg accagcaata gcgtgttctg ggcagaaggc
     2041 ggggcgaaag ttcagctgaa tggtagtggt ctgaccgtac aggcatcccc gctctccaga
     2101 gcattaaagg gagccattag cttcgacaac ctcagcggtg ccagcgccag tcagcgtaaa
     2161 ggcgataagc gtattctgta tgcttccgaa acagcggccc gtgcggttgg cgggcagatt
     2221 acgcttcacg ctttcgatgc cggaaaactg gcggtcggga tgccaattcg ctatctcggt
     2281 attgatatcg ggcaaatcca gacgctggat ctgattaccg cacgcaatga agtacaggca
     2341 aaggcggtac tttacccgga atatgtccag acctttgctc gcggtggtac gcgcttctca
     2401 gtggtcacac cgcaaatttc ggcagctggc gttgagcatc ttgatactat cctccagccg
     2461 tatatcaacg tcgaaccagg ccggggcaat cctcgccgcg actttgaatt acaagaggcc
     2521 accattactg attcgcgtta cctggatggc ttaagcatta ttgttgaagc gccggaagcc
     2581 ggttcgttag gcatcggtac gcctgtgctg ttccgtggtc tggaagtcgg tacggttaca
     2641 ggaatgacgc tggggacatt gtcagatcgc gtgatgattg cgatgcgcat cagtaaacgc
     2701 tatcaacacc tggtgcgtaa caattccgtc ttctggttgg catcgggtta cagtctggac
     2761 tttggtctga cgggcggcgt agtgaaaacc ggcaccttta accagtttat ccgtggcggc
     2821 atcgccttcg ccacgcctcc gggtacgcca ctggcaccga aagcccagga aggcaaacac
     2881 ttcctgttgc aggaaagtga accgaaagag tggcgtgaat ggggaactgc gcttcccaaa
     2941 ggcgagaccc atcaccacca tcaccattaa ggccaggccg gccgatccgg ctgctaacaa
     3001 agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag cataacccct
     3061 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt
     3121 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     3181 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     3241 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     3301 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     3361 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     3421 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     3481 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     3541 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc
     3601 ggggaacgac gacctgcagc caagctagct tggctggacg taaactcctc ttcagaccta
     3661 ataacttcgt atagcataca ttatacgaag ttatattaag ggttattgaa tatgatcgga
     3721 attgggctgc aggaattcga tagcttggct gcaggtcgac ctcgaggatg tgcgcggaac
     3781 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     3841 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     3901 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     3961 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     4021 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     4081 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     4141 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     4201 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     4261 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     4321 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     4381 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt
     4441 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     4501 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     4561 tattgctgat aaatctggag ccggtgagcg tggctctcgc ggtatcattg cagcactggg
     4621 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     4681 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     4741 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     4801 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     4861 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     4921 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     4981 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     5041 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     5101 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     5161 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     5221 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     5281 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     5341 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     5401 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     5461 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     5521 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     5581 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     5641 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     5701 ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct
     5761 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     5821 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     5881 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     5941 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     6001 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     6061 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     6121 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     6181 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     6241 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     6301 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     6361 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     6421 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     6481 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     6541 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga
     6601 taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga
     6661 gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa
     6721 agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga
     6781 taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc
     6841 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     6901 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     6961 agctgcatta atgaatcggc caacgcgcgg ggaga
//
