LOCUS       sequence-309620-        7346 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for naturally occurring 5xMCE protein (without TM
            region).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7346)
  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 7346)
  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: pBEL1752
FEATURES             Location/Qualifiers
     source          1..7346
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     87..106
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     190..209
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        190..208
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    209..233
                     /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             2093..2110
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2176..2194)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2190..2237
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2274..2729
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2361..2380)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2571..2592
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(2804..2837)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2916..3020
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3021..3089,3090..3881)
                     /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(3239..3258)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4052..4640
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4541..4560
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4794..4811
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4826..4965
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4891..4910)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5051..5073
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5067..5258)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6067..7149)
                     /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     7111..7130
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7150..7227)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
       61 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
      121 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
      181 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
      241 aaataatttt gtttaacttt ggcccaggcg gccaggaggg tctcacatgg gcatcatgat
      301 ttctgttgtc ttcgattccg cagaagggtt agaagccggc aaaacgcgcg ttatgtacaa
      361 aggccttccc gcaggcgaaa tccgtcaatt aacacttaat gaagatttgc agagtattac
      421 tgccttaatt gagatgaata tcaacactga ggatatgttg actgataata cgcttttctg
      481 gctggtaaaa cctcaagtta gtctgtccgg ggtatctggt ctggagacac ttatctccgg
      541 ttactacatt gggattcaac cggcggaggg agaatcgaag cgcagtttca cagctctgac
      601 gtcgccacct ccaccagaac gcgatgagca agggttatac attacgttgt acacggattc
      661 tgcccgttca attcagactg ggtccccagt ttacttccgt aatattgaag ccggtcgcgt
      721 aatcgagtac tccttgtccc ctagtggcga ggaggttttg attgatgtat atatccagcc
      781 ggagtttaca gcgtgggtaa ctgaacgctc gcgcttttgg aacgcgtcag gcattgagat
      841 tgattcgaat ttgcctgatt tttcagtccg cgtgggttcg ctgatgtcga ttttaaccgg
      901 aggtatccat ttcgactccc ctgcctttgg aactccaaaa gctgcagtta atggcgaccg
      961 cttcccgttg tatccggacc ttgagagctc ccgcgacgat ctggcttatc agcccgcggg
     1021 atttgaaggg attagttttc gcttacgcgc tgacagcttg gggagcattc agagcgggtc
     1081 cccgattctg tttaaaggca ttaaagtagg tcaggtcacc ggctatcaac tgaatactag
     1141 tgatcatagt gtgacgattt acgcctcctt gcgccaacgc tattcatccc ttgtcaagcg
     1201 cgattcgcgt ttttggaatg taagtgggat ccgcagtagt tttaatttgc aaagcggctt
     1261 cagcttcgaa accgatggtt tgaagcccct gatcagtggg ggcattgcgt tcgacacgac
     1321 ggatggtcca tcggcacagg cggggcaaat ctttacttta ttcaactcgg aggcagagtc
     1381 actgagcaat actcttgcaa tcactcttga atttccgcca ggaccatctt tacttgcagg
     1441 gagccaaatt cgctttcgca accaacccgt gggcgaaatc gtgtcggtgg agctttcagg
     1501 gagtgacggc tcgttgtttg ctcgtgcctt gctttttgag caaggctttt tcttggcacg
     1561 tcaggggaca cgtttttgga ttgagacgcc tcaaattgga ttgagcggta tccagaaccc
     1621 caccggtttg gtgtttggca accacgtatc cgttgcgcct ggattggggg agaactcgca
     1681 aactcatttt gtggcattga ctgaagctcc agttttccag ccacaaccgg gcttgaacct
     1741 tgtccttact gccccctccc tggggagctt aaatcagaat agtaaacttt tttaccgcgg
     1801 aatctccgta ggcaaggtca tcgggtttca gttgagccgc gaaggccacc gtgtagaaat
     1861 ctatgtcaac atcagccccc gttaccagca cctgattacc caatcgactc aattccgtaa
     1921 cattggtgga atccgtgcct cagcatcatt gtttaaagga gtcgatatcg acgccccgtc
     1981 tatcgaagca ttattatcgg ggggtattga aatcgaaatt ccctacgaag atgcccccgc
     2041 aacggaaggc ttacacttcc ccttacaacc cgcgagccgc caaggcgaga cccatcacca
     2101 ccatcaccat taaggccagg ccggccgatc cggctgctaa caaagcccga aaggaagctg
     2161 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     2221 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc
     2281 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2341 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2401 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2461 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2521 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     2581 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     2641 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     2701 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaac gacgacctgc
     2761 agccaagcta gcttggctgg acgtaaactc ctcttcagac ctaataactt cgtatagcat
     2821 acattatacg aagttatatt aagggttatt gaatatgatc ggaattgggc tgcaggaatt
     2881 cgatagcttg gctgcaggtc gacctcgagg atgtgcgcgg aacccctatt tgtttatttt
     2941 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     3001 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     3061 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     3121 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     3181 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     3241 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     3301 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     3361 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     3421 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     3481 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     3541 acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg
     3601 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     3661 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     3721 gagccggtga gcgtggctct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     3781 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     3841 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     3901 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     3961 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     4021 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     4081 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     4141 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     4201 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     4261 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     4321 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     4381 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     4441 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     4501 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     4561 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     4621 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     4681 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     4741 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     4801 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     4861 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     4921 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     4981 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     5041 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     5101 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     5161 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     5221 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     5281 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     5341 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     5401 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     5461 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     5521 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     5581 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     5641 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     5701 aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg
     5761 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     5821 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa
     5881 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     5941 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     6001 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     6061 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     6121 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     6181 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     6241 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     6301 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     6361 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     6421 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     6481 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     6541 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     6601 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     6661 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     6721 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     6781 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     6841 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     6901 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     6961 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     7021 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     7081 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     7141 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     7201 aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct
     7261 gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg
     7321 gtgcatgcaa ggagatggcg cccaac
//
