LOCUS       sequence-342013-        7718 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for LetAB with 6 ring (rings 123457) LetB 
            (without TM region) mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7718)
  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 7718)
  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: pBEL2004
FEATURES             Location/Qualifiers
     source          1..7718
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     61..78
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    93..232
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(158..177)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     318..340
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(334..525)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1334..2416)
                     /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     2378..2397
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2417..2494)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2700..2719
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2803..2822
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2803..2821
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2822..2846
                     /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             5078..5095
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5161..5179)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5175..5222
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5259..5714
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5346..5365)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5556..5577
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(5789..5822)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        5901..6005
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6006..6074,6075..6866)
                     /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(6224..6243)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7037..7625
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7526..7545
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
       61 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
      121 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
      181 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
      241 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
      301 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
      361 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
      421 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
      481 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
      541 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
      601 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
      661 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
      721 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
      781 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
      841 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
      901 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
      961 caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg
     1021 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
     1081 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
     1141 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac
     1201 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     1261 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     1321 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     1381 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     1441 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     1501 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     1561 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg
     1621 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     1681 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     1741 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     1801 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     1861 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     1921 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
     1981 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
     2041 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
     2101 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
     2161 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
     2221 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
     2281 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
     2341 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
     2401 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
     2461 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc
     2521 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
     2581 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
     2641 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
     2701 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
     2761 atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag
     2821 gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac tttggcccag
     2881 gcggccagga gggtctcaca tgcaggaccg gggtaatacc gtcaccatcg actttatgtc
     2941 ggcggatggt attgttccag gccgtacgcc tgttcgttat cagggcgttg aagtcggaac
     3001 agtgcaggat atcagcctca gcgacgatct tcgtaagatt gaagtcaagg tcagcatcaa
     3061 gtccgatatg aaagatgcgc tgcgcgaaga gactcagttc tggctggtga cgccaaaagc
     3121 atcgttggca ggtgtctccg ggctggacgc cctcgtcggt gggaactata tcggcatgat
     3181 gccgggtaaa ggtaaagagc aggatcactt tgtcgcactc gatacccaac cgaaatatcg
     3241 gctggacaat ggcgatctga tgatccacct gcaagccccc gatctcggtt cgctgaacag
     3301 cggttcattg gtctatttcc gcaagatccc ggtgggaaaa gtctacgact atgccatcaa
     3361 tcccaacaag caaggcgtgg tgattgatgt cctgatcgag cggcgtttta ccgatctggt
     3421 gaaaaaaggt agccgtttct ggaacgtttc cggcgttgat gccaacgtca gtatcagtgg
     3481 cgcgaaggtg aaactggaaa gcctggcggc actggttaac ggtgcgattg ccttcgattc
     3541 accagaagag tcgaaacctg ccgaggcgga agataccttt ggtctgtatg aagatctggc
     3601 ccacagccag cgtggcgtaa taataaaact ggaactgccg agtggggccg gattaaccgc
     3661 cgactcgacg ccgttaatgt atcaggggct ggaagtcgga cagctgacta aactggattt
     3721 aaatcctggt ggtaaagtca caggggaaat gaccgttgat cccagcgtcg ttaccctgct
     3781 tcgtgaaaat acccgcatcg aattacgcaa cccgaaatta tcccttagcg atgctaatct
     3841 cagcgccctg ctgaccggca aaaccttcga gctggtgccc ggcgatggcg agccacgcaa
     3901 agagttcgtt gttgtgccag gcgaaaaagc actgctgcat gaacctgatg ttctgacgct
     3961 gaccctgacc gcgccggaaa gttacggtat tgatgcgggt cagccgctca ttcttcacgg
     4021 cgtgcaggta ggccaggtta tcgatcgtaa actcaccagc aaaggcgtca cctttaccgt
     4081 cgccatcgag cctcagcatc gcgaactggt aaaaggcgat agcaaatttg tcgtcaacag
     4141 ccgtgtcgat gtgaaggtgg ggctggatgg cgttgagttt ctcggtgcca gcgcctcaga
     4201 atggatcaat ggcgggatac gtattctgcc gggcgataaa ggtgagatga aagccagcta
     4261 tccactgtat gccaatttgg aaaaagcgct ggagaacagc cttagcgatt tacccaccac
     4321 aaccgtgagt ttgagtgcag agacgctgcc ggatgtgcag gcaggatcgg tagtgctgta
     4381 ccgtaaattt gaagttggtg aagtgattac cgtgcgtccg cgagctaacg cgtttgatat
     4441 cgatctgcat attaagccgg agtatcgcaa ccttctgacc agcaatagcg tgttctgggc
     4501 agaaggcggg gcgaaagttc agctgaatgg tagtggtctg accgtacagg catccccgct
     4561 ctccagagca ttaaagggag ccattagctt cgacaacctc agcggtgcca gcgccagtca
     4621 gcgtaaaggc gataagcgta ttctgtatgc ttccgaaaca gcggcccgtg cggttggctt
     4681 aagcattatt gttgaagcgc cggaagccgg ttcgttaggc atcggtacgc ctgtgctgtt
     4741 ccgtggtctg gaagtcggta cggttacagg aatgacgctg gggacattgt cagatcgcgt
     4801 gatgattgcg atgcgcatca gtaaacgcta tcaacacctg gtgcgtaaca attccgtctt
     4861 ctggttggca tcgggttaca gtctggactt tggtctgacg ggcggcgtag tgaaaaccgg
     4921 cacctttaac cagtttatcc gtggcggcat cgccttcgcc acgcctccgg gtacgccact
     4981 ggcaccgaaa gcccaggaag gcaaacactt cctgttgcag gaaagtgaac cgaaagagtg
     5041 gcgtgaatgg ggaactgcgc ttcccaaagg cgagacccat caccaccatc accattaagg
     5101 ccaggccggc cgatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
     5161 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     5221 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
     5281 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     5341 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     5401 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     5461 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     5521 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     5581 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     5641 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     5701 aacgcttaca atttaggtgg cacttttcgg ggaacgacga cctgcagcca agctagcttg
     5761 gctggacgta aactcctctt cagacctaat aacttcgtat agcatacatt atacgaagtt
     5821 atattaaggg ttattgaata tgatcggaat tgggctgcag gaattcgata gcttggctgc
     5881 aggtcgacct cgaggatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     5941 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     6001 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     6061 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     6121 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     6181 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     6241 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     6301 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     6361 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     6421 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     6481 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     6541 cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     6601 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     6661 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     6721 gctctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     6781 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     6841 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     6901 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     6961 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     7021 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     7081 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     7141 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     7201 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     7261 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     7321 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     7381 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     7441 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     7501 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     7561 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     7621 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     7681 acatgttctt tcctgcgtta tcccctgatt ctgtggat
//
