LOCUS       sequence-309715-        7361 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for LetAB with 5 ring (rings 12347) LetB (without
            TM region) mutant .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7361)
  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 7361)
  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: pBEL2003
FEATURES             Location/Qualifiers
     source          1..7361
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     970..989
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1009..1086)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1292..1311
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1395..1414
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1395..1413
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1414..1438
                     /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             3313..3330
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3396..3414)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3410..3457
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3494..3949
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3581..3600)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3791..3812
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(4024..4057)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4136..4240
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4241..4309,4310..5101)
                     /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(4459..4478)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5272..5860
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5761..5780
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6014..6031
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6046..6185
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6111..6130)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6271..6293
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6287..6478)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7287..7361,1..1008))
                     /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 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
       61 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
      121 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
      181 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
      241 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
      301 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
      361 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
      421 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
      481 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
      541 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
      601 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
      661 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
      721 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
      781 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
      841 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
      901 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
      961 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     1021 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     1081 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     1141 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     1201 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     1261 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     1321 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     1381 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     1441 tctagaaata attttgttta actttggccc aggcggccag gagggtctca catgcaggac
     1501 cggggtaata ccgtcaccat cgactttatg tcggcggatg gtattgttcc aggccgtacg
     1561 cctgttcgtt atcagggcgt tgaagtcgga acagtgcagg atatcagcct cagcgacgat
     1621 cttcgtaaga ttgaagtcaa ggtcagcatc aagtccgata tgaaagatgc gctgcgcgaa
     1681 gagactcagt tctggctggt gacgccaaaa gcatcgttgg caggtgtctc cgggctggac
     1741 gccctcgtcg gtgggaacta tatcggcatg atgccgggta aaggtaaaga gcaggatcac
     1801 tttgtcgcac tcgataccca accgaaatat cggctggaca atggcgatct gatgatccac
     1861 ctgcaagccc ccgatctcgg ttcgctgaac agcggttcat tggtctattt ccgcaagatc
     1921 ccggtgggaa aagtctacga ctatgccatc aatcccaaca agcaaggcgt ggtgattgat
     1981 gtcctgatcg agcggcgttt taccgatctg gtgaaaaaag gtagccgttt ctggaacgtt
     2041 tccggcgttg atgccaacgt cagtatcagt ggcgcgaagg tgaaactgga aagcctggcg
     2101 gcactggtta acggtgcgat tgccttcgat tcaccagaag agtcgaaacc tgccgaggcg
     2161 gaagatacct ttggtctgta tgaagatctg gcccacagcc agcgtggcgt aataataaaa
     2221 ctggaactgc cgagtggggc cggattaacc gccgactcga cgccgttaat gtatcagggg
     2281 ctggaagtcg gacagctgac taaactggat ttaaatcctg gtggtaaagt cacaggggaa
     2341 atgaccgttg atcccagcgt cgttaccctg cttcgtgaaa atacccgcat cgaattacgc
     2401 aacccgaaat tatcccttag cgatgctaat ctcagcgccc tgctgaccgg caaaaccttc
     2461 gagctggtgc ccggcgatgg cgagccacgc aaagagttcg ttgttgtgcc aggcgaaaaa
     2521 gcactgctgc atgaacctga tgttctgacg ctgaccctga ccgcgccgga aagttacggt
     2581 attgatgcgg gtcagccgct cattcttcac ggcgtgcagg taggccaggt tatcgatcgt
     2641 aaactcacca gcaaaggcgt cacctttacc gtcgccatcg agcctcagca tcgcgaactg
     2701 gtaaaaggcg atagcaaatt tgtcgtcaac agccgtgtcg atgtgaaggt ggggctggat
     2761 ggcgttgagt ttctcggtgc cagcgcctca gaatggatca atggcgggat acgtattctg
     2821 ccgggcgata aaggtgagat gaaagccagc tatccactgt atgccaattt ggaaaaagcg
     2881 ctggagaaca gccttagcga tttacccacc ggcttaagca ttattgttga agcgccggaa
     2941 gccggttcgt taggcatcgg tacgcctgtg ctgttccgtg gtctggaagt cggtacggtt
     3001 acaggaatga cgctggggac attgtcagat cgcgtgatga ttgcgatgcg catcagtaaa
     3061 cgctatcaac acctggtgcg taacaattcc gtcttctggt tggcatcggg ttacagtctg
     3121 gactttggtc tgacgggcgg cgtagtgaaa accggcacct ttaaccagtt tatccgtggc
     3181 ggcatcgcct tcgccacgcc tccgggtacg ccactggcac cgaaagccca ggaaggcaaa
     3241 cacttcctgt tgcaggaaag tgaaccgaaa gagtggcgtg aatggggaac tgcgcttccc
     3301 aaaggcgaga cccatcacca ccatcaccat taaggccagg ccggccgatc cggctgctaa
     3361 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     3421 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     3481 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3541 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3601 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3661 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     3721 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3781 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     3841 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     3901 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     3961 ttcggggaac gacgacctgc agccaagcta gcttggctgg acgtaaactc ctcttcagac
     4021 ctaataactt cgtatagcat acattatacg aagttatatt aagggttatt gaatatgatc
     4081 ggaattgggc tgcaggaatt cgatagcttg gctgcaggtc gacctcgagg atgtgcgcgg
     4141 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     4201 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     4261 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     4321 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     4381 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     4441 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     4501 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     4561 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     4621 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     4681 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     4741 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac
     4801 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     4861 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     4921 gtttattgct gataaatctg gagccggtga gcgtggctct cgcggtatca ttgcagcact
     4981 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     5041 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     5101 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     5161 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     5221 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     5281 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     5341 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     5401 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     5461 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     5521 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     5581 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     5641 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     5701 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     5761 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     5821 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     5881 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     5941 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     6001 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     6061 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
     6121 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     6181 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     6241 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     6301 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     6361 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     6421 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     6481 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     6541 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     6601 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     6661 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     6721 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     6781 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     6841 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     6901 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg
     6961 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     7021 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     7081 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     7141 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     7201 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     7261 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     7321 gccagctgca ttaatgaatc ggccaacgcg cggggagagg c
//
