LOCUS       sequence-270776-        8031 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for LetB PLL4 deletion (without TM region).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8031)
  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 8031)
  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: pBEL1996
FEATURES             Location/Qualifiers
     source          1..8031
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      12..467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(99..118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     309..330
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(543..576)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        655..759
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(760..828,829..1620)
                     /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(978..997)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1791..2379
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2280..2299
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2533..2550
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2565..2707
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2633..2652)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2793..2815
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2809..3000)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3809..4891)
                     /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     4853..4872
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4892..4969)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5175..5194
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5278..5297
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5278..5296
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5297..5321
                     /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             7862..7879
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(7945..7963)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7959..8006
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt
      481 tcggggaacg acgacctgca gccaagctag cttggctgga cgtaaactcc tcttcagacc
      541 taataacttc gtatagcata cattatacga agttatatta agggttattg aatatgatcg
      601 gaattgggct gcaggaattc gatagcttgg ctgcaggtcg acctcgagga tgtgcgcgga
      661 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
      721 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
      781 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
      841 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
      901 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
      961 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     1021 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     1081 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     1141 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     1201 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     1261 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg
     1321 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     1381 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     1441 tttattgctg ataaatctgg agccggtgag cgtggctctc gcggtatcat tgcagcactg
     1501 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     1561 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     1621 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     1681 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     1741 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     1801 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     1861 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     1921 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     1981 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     2041 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     2101 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     2161 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     2221 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     2281 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     2341 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     2401 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     2461 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     2521 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     2581 ctccttacgc atctgtgcgg tatttcacac cgcatatatg gtgcactctc agtacaatct
     2641 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     2701 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     2761 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     2821 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     2881 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     2941 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     3001 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     3061 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     3121 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     3181 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     3241 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     3301 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     3361 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     3421 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc
     3481 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     3541 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     3601 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     3661 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     3721 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     3781 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     3841 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3901 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     3961 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     4021 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     4081 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     4141 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     4201 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     4261 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     4321 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     4381 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     4441 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     4501 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     4561 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     4621 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     4681 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     4741 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     4801 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     4861 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     4921 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     4981 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     5041 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     5101 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     5161 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     5221 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa
     5281 tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt
     5341 ttaactttgg cccaggcggc caggagggtc tcacatgcag gaccggggta ataccgtcac
     5401 catcgacttt atgtcggcgg atggtattgt tccaggccgt acgcctgttc gttatcaggg
     5461 cgttgaagtc ggaacagtgc aggacatcag cctcagcgac gatcttcgta agattgaagt
     5521 caaggtcagc atcaagtccg atatgaaaga tgcgctgcgc gaagagactc agttctggct
     5581 ggtgacgcca aaagcatcgt tggcaggtgt ctccgggctg gacgccctcg tcggtgggaa
     5641 ctatatcggc atgatgcccg ggaaaggtaa agagcaggat cactttgtcg cactcgatac
     5701 ccaaccgaaa tatcggctgg acaatggcga tctgatgatc cacctgcaag cccccgatct
     5761 cggttcgctg aacagcggtt cattggtcta tttccgcaag atcccggtgg gaaaagtcta
     5821 cgactatgcc atcaatccca acaagcaagg cgtggtgatt gatgtcctga tcgagcggcg
     5881 ttttaccgat ctggtgaaaa aaggtagccg tttctggaac gtttccggcg ttgatgccaa
     5941 cgtcagtatc agtggcgcga aggtgaaact ggaaagcttg gcggcactgg ttaacggtgc
     6001 gattgccttc gattcaccag aagagtcgaa acctgccgag gcggaagata cctttggtct
     6061 gtatgaagat ctggcccaca gccagcgtgg cgtaataata aaactggaac tgccgagtgg
     6121 ggccggatta accgccgact cgacgccgtt aatgtatcag gggctggaag tcggacagct
     6181 gactaaactg gatttaaatc ctggtggtaa agtcacaggg gaaatgaccg ttgatcccag
     6241 cgtcgttacc ctgcttcgtg aaaatacccg catcgaatta cgcaacccga aattatccct
     6301 tagcgatgct aatctcagcg ccctgctgac cggcaaaacc ttcgagctgg tgcccggcga
     6361 tggcgagcca cgcaaagaat tcgttgttgt gccaggcgaa aaagcactgc tgcatgaacc
     6421 tgatgttctg acgctgaccc tgaccgcgcc ggaaagttac ggtattgatg cgggtcagcc
     6481 gctcattctt cacggcgtgc aggtaggcca ggttatcgat cgtaaactca ccagcaaagg
     6541 cgtcaccttt accgtcgcca tcgagcctca gcatcgcgaa ctggtaaaag gcgatagcaa
     6601 atttgtcgtc aacagccgtg tcgatctcgg tgccagcgcc tcagaatgga tcaatggcgg
     6661 gatccgtatt ctgccgggcg ataaaggtga gatgaaagcc agctatccac tgtatgccaa
     6721 tttggaaaaa gcgctggaga acagccttag cgatttaccc accacaaccg tgagtttgag
     6781 tgcagagacg ctgccggatg tgcaggcagg atcggtagtg ctgtaccgta aatttgaagt
     6841 tggtgaagtg attaccgtgc gtccgcgagc taacgcgttt gacatcgatc tgcatattaa
     6901 gccggagtat cgcaaccttc tgaccagcaa tagcgtgttc tgggcagaag gcggggcgaa
     6961 agttcagctg aatggtagtg gtctgaccgt acaggcatcc ccgctctcca gagctctaaa
     7021 gggagccatt agcttcgaca acctcagcgg tgccagcgcc agtcagcgta aaggcgataa
     7081 gcgtattctg tatgcttccg aaacagcggc ccgtgcggtt ggcgggcaga ttacgcttca
     7141 cgctttcgat gccggaaaac tggcggtcgg gatgccaatt cgctatctcg gtattgacat
     7201 cgggcaaatc cagacgctgg atctgattac cgcacgcaat gaagtacagg caaaggcggt
     7261 actttacccg gaatatgtcc agacctttgc tcgcggtggt acgcgcttct cagtggtcac
     7321 accgcaaatt tcggcagctg gcgttgagca tcttgatact atcctccagc cgtatatcaa
     7381 cgtcgaacca ggccggggca atcctcgccg cgactttgaa ttacaagagg ccaccattac
     7441 tgattcgcgg tacctggatg gcttaagcat tattgttgaa gcgccggaag ccggttcgtt
     7501 aggcatcggt acgcctgtgc tgttccgtgg tctggaagtc ggtacggtta caggaatgac
     7561 gctggggaca ttgtcagatc gcgtgatgat tgcgatgcgc atcagtaaac gctatcaaca
     7621 cctggtgcgt aacaattccg tcttctggtt ggcatcgggt tacagtctgg actttggtct
     7681 gacgggcggc gtagtgaaaa ccggcacctt taaccagttt atccgtggcg gcatcgcctt
     7741 cgccacgcct ccgggtacgc cactggcacc gaaagcccag gaaggcaaac acttcctgtt
     7801 gcaggaaagt gaaccgaaag agtggcgtga atggggaact gcgcttccca aaggcgagac
     7861 ccatcaccac catcaccatt aaggccaggc cggccgatcc ggctgctaac aaagcccgaa
     7921 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     7981 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga t
//
