LOCUS       sequence-270762-        6639 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for LetAB with 3 ring (rings 167) LetB (without 
            TM region) mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6639)
  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 6639)
  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: pBEL2001
FEATURES             Location/Qualifiers
     source          1..6639
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     34..51
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    66..208
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(134..153)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     294..316
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(310..501)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1310..2392)
                     /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     2354..2373
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2393..2470)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2676..2695
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2779..2798
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2779..2797
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2798..2822
                     /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(4683..4716)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4795..4899
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4900..4968,4969..5760)
                     /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(5118..5137)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5931..6519
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6420..6439
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
       61 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatat
      121 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
      181 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
      241 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
      301 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
      361 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
      421 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
      481 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
      541 aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc
      601 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
      661 aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg
      721 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
      781 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
      841 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
      901 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg
      961 cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
     1021 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
     1081 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
     1141 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
     1201 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     1261 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     1321 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     1381 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     1441 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     1501 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     1561 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
     1621 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     1681 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     1741 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     1801 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     1861 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     1921 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     1981 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     2041 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     2101 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     2161 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     2221 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     2281 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     2341 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     2401 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     2461 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc
     2521 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg
     2581 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
     2641 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
     2701 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag
     2761 atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt
     2821 cccctctaga aataattttg tttaactttg gcccaggcgg ccaggagggt ctcacatgca
     2881 ggaccggggt aataccgtca ccatcgactt tatgtcggcg gatggtattg ttccaggccg
     2941 tacgcctgtt cgttatcagg gcgttgaagt cggaacagtg caggatatca gcctcagcga
     3001 cgatcttcgt aagattgaag tcaaggtcag catcaagtcc gatatgaaag atgcgctgcg
     3061 cgaagagact cagttctggc tggtgacgcc aaaagcatcg ttggcaggtg tctccgggct
     3121 ggacgccctc gtcggtggga actatatcgg catgatgccg ggtaaaggta aagagcagga
     3181 tcactttgtc gcactcgata cccaaccgaa atatcggctg gacaatggcg atgggcagat
     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 aaagcccagg aaggcaaaca
     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 attaacgttt acaatttcag gtggcacttt
     4621 tcggggaacg acgacctgca gccaagctag cttggctgga cgtaaactcc tcttcagacc
     4681 taataacttc gtatagcata cattatacga agttatatta agggttattg aatatgatcg
     4741 gaattgggct gcaggaattc gatagcttgg ctgcaggtcg acctcgagga tgtgcgcgga
     4801 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     4861 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     4921 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     4981 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     5041 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     5101 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     5161 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     5221 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     5281 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     5341 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     5401 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg
     5461 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     5521 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     5581 tttattgctg ataaatctgg agccggtgag cgtggctctc gcggtatcat tgcagcactg
     5641 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     5701 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     5761 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     5821 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     5881 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     5941 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     6001 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     6061 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     6121 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     6181 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     6241 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     6301 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     6361 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     6421 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     6481 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     6541 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     6601 gattctgtgg ataaccgtat taccgccttt gagtgagct
//
