LOCUS       sequence-309744-        8027 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 8027)
  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 8027)
  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..8027
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     153..175
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(169..360)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1169..2251)
                     /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     2213..2232
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2252..2329)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2535..2554
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2638..2657
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2638..2656
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2657..2681
                     /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             5222..5239
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5305..5323)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5319..5366
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5403..5858
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5490..5509)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5700..5721
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(5933..5966)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        6045..6149
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6150..6218,6219..7010)
                     /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(6368..6387)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7181..7769
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7670..7689
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7923..7940
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(7955..8027,1..67)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(join(8020..8027,1..12))
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
       61 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
      121 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
      181 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
      241 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
      301 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
      361 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
      421 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
      481 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
      541 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
      601 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
      661 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
      721 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
      781 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc
      841 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
      901 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
      961 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     1021 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     1081 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     1141 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     1201 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     1261 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     1321 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     1381 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     1441 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     1501 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     1561 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     1621 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     1681 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     1741 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     1801 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     1861 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     1921 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     1981 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     2041 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     2101 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     2161 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     2221 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     2281 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     2341 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     2401 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     2461 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     2521 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     2581 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa
     2641 tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt
     2701 ttaactttgg cccaggcggc caggagggtc tcacatgcag gaccggggta ataccgtcac
     2761 catcgacttt atgtcggcgg atggtattgt tccaggccgt acgcctgttc gttatcaggg
     2821 cgttgaagtc ggaacagtgc aggacatcag cctcagcgac gatcttcgta agattgaagt
     2881 caaggtcagc atcaagtccg atatgaaaga tgcgctgcgc gaagagactc agttctggct
     2941 ggtgacgcca aaagcatcgt tggcaggtgt ctccgggctg gacgccctcg tcggtgggaa
     3001 ctatatcggc atgatgcccg ggaaaggtaa agagcaggat cactttgtcg cactcgatac
     3061 ccaaccgaaa tatcggctgg acaatggcga tctgatgatc cacctgcaag cccccgatct
     3121 cggttcgctg aacagcggtt cattggtcta tttccgcaag atcccggtgg gaaaagtcta
     3181 cgactatgcc atcaatccca acaagcaagg cgtggtgatt gatgtcctga tcgagcggcg
     3241 ttttaccgat ctggtgaaaa aaggtagccg tttctggaac gtttccggcg ttgatgccaa
     3301 cgtcagtatc agtggcgcga aggtgaaact ggaaagcttg gcggcactgg ttaacggtgc
     3361 gattgccttc gattcaccag aagagtcgaa acctgccgag gcggaagata cctttggtct
     3421 gtatgaagat ctggcccaca gccagcgtgg cgtaataata aaactggaac tgccgagtgg
     3481 ggccggatta accgccgact cgacgccgtt aatgtatcag gggctggaag tcggacagct
     3541 gactaaactg gatttaaatc ctggtggtaa agtcacaggg gaaatgaccg ttgatcccag
     3601 cgtcgttacc ctgcttcgtg aaaatacccg catcgaatta cgcaacccga aattatccct
     3661 tagcgatgct aatctcagcg ccctgctgac cggcaaaacc ttcgagctgg tgcccggcga
     3721 tggcgagcca cgcaaagaat tcgttgttgt gccaggcgaa aaagcactgc tgcatgaacc
     3781 tgatgttctg acgctgaccc tgaccgcgcc ggaaagttac ggtattgatg cgggtcagcc
     3841 gctcattctt cacggcgtgc aggtaggcca ggttatcgat cgtaaactca ccagcaaagg
     3901 cgtcaccttt accgtcgcca tcgagcctca gcatcgcgaa ctggtaaaag gcgatagcaa
     3961 atttgtcgtc aacagccgtg tcgatctcgg tgccagcgcc tcagaatgga tcaatggcgg
     4021 gatccgtatt ctgccgggcg ataaaggtga gatgaaagcc agctatccac tgtatgccaa
     4081 tttggaaaaa gcgctggaga acagccttag cgatttaccc accacaaccg tgagtttgag
     4141 tgcagagacg ctgccggatg tgcaggcagg atcggtagtg ctgtaccgta aatttgaagt
     4201 tggtgaagtg attaccgtgc gtccgcgagc taacgcgttt gacatcgatc tgcatattaa
     4261 gccggagtat cgcaaccttc tgaccagcaa tagcgtgttc tgggcagaag gcggggcgaa
     4321 agttcagctg aatggtagtg gtctgaccgt acaggcatcc ccgctctcca gagctctaaa
     4381 gggagccatt agcttcgaca acctcagcgg tgccagcgcc agtcagcgta aaggcgataa
     4441 gcgtattctg tatgcttccg aaacagcggc ccgtgcggtt ggcgggcaga ttacgcttca
     4501 cgctttcgat gccggaaaac tggcggtcgg gatgccaatt cgctatctcg gtattgacat
     4561 cgggcaaatc cagacgctgg atctgattac cgcacgcaat gaagtacagg caaaggcggt
     4621 actttacccg gaatatgtcc agacctttgc tcgcggtggt acgcgcttct cagtggtcac
     4681 accgcaaatt tcggcagctg gcgttgagca tcttgatact atcctccagc cgtatatcaa
     4741 cgtcgaacca ggccggggca atcctcgccg cgactttgaa ttacaagagg ccaccattac
     4801 tgattcgcgg tacctggatg gcttaagcat tattgttgaa gcgccggaag ccggttcgtt
     4861 aggcatcggt acgcctgtgc tgttccgtgg tctggaagtc ggtacggtta caggaatgac
     4921 gctggggaca ttgtcagatc gcgtgatgat tgcgatgcgc atcagtaaac gctatcaaca
     4981 cctggtgcgt aacaattccg tcttctggtt ggcatcgggt tacagtctgg actttggtct
     5041 gacgggcggc gtagtgaaaa ccggcacctt taaccagttt atccgtggcg gcatcgcctt
     5101 cgccacgcct ccgggtacgc cactggcacc gaaagcccag gaaggcaaac acttcctgtt
     5161 gcaggaaagt gaaccgaaag agtggcgtga atggggaact gcgcttccca aaggcgagac
     5221 ccatcaccac catcaccatt aaggccaggc cggccgatcc ggctgctaac aaagcccgaa
     5281 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     5341 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
     5401 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     5461 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     5521 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     5581 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     5641 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     5701 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     5761 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     5821 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaacg
     5881 acgacctgca gccaagctag cttggctgga cgtaaactcc tcttcagacc taataacttc
     5941 gtatagcata cattatacga agttatatta agggttattg aatatgatcg gaattgggct
     6001 gcaggaattc gatagcttgg ctgcaggtcg acctcgagga tgtgcgcgga acccctattt
     6061 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     6121 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     6181 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     6241 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     6301 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     6361 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     6421 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     6481 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     6541 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     6601 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     6661 taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac
     6721 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     6781 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     6841 ataaatctgg agccggtgag cgtggctctc gcggtatcat tgcagcactg gggccagatg
     6901 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     6961 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     7021 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     7081 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     7141 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     7201 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     7261 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     7321 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     7381 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     7441 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     7501 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     7561 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     7621 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     7681 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     7741 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     7801 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     7861 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     7921 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     7981 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatct
//
