LOCUS       sequence-309619-        7040 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for naturally occurring 4xMCE protein (without TM
            region).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7040)
  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 7040)
  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: pBEL1751
FEATURES             Location/Qualifiers
     source          1..7040
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     88..107
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     191..210
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        191..209
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    210..234
                     /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             1788..1805
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1871..1889)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1885..1932
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1969..2424
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2056..2075)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2266..2287
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(2499..2532)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2611..2715
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2716..2784,2785..3576)
                     /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(2934..2953)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3747..4335
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4236..4255
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4489..4506
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4521..4660
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4586..4605)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4746..4768
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4762..4953)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5762..6844)
                     /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     6806..6825
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6845..6922)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
       61 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
      121 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat
      181 cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta
      241 gaaataattt tgtttaactt tggcccaggc ggccaggagg gtctcacatg ggcattatga
      301 taaccgtcgc aatcaaagat gccaatgggc tgactgaggg gaagacccaa gtatttttca
      361 aaggattgcc agttgggacc ttgaaggggt tcactgtttc ccccgatctt aaacatatca
      421 acgcggagat tgaaatggtg aagcaggctc gcgagaaact gactaaagac acattattct
      481 gggttgtacg tccggagatt tccatgaacc gcatcacggg tttggacacc ttaattaagg
      541 ggagttactt cgaggtgcag cctggtaatt cgacagagca aacagactca tttacggcat
      601 tgccggaccc accccctatt tctatgcgtg tgccaggatt gcacttgaca ctgcacaata
      661 aagactcagt gtcgctggaa gcaggaagcc cagtgtcctt caaaaagatc gctgtaggtg
      721 agatcgtgtc taatcgttta cttcctgatg gaacgattga aactaagctt ttgattcacc
      781 ctcaatatgt gggccatgtc acaagtttct ctcgtttttt tatctcatct ggaatccgtt
      841 tccaggcgaa cttaccgaag atcagcatgg aggttgactc aattaaatca atcttgcgcg
      901 ggggtattag tttcttcacg cccacgggcg gcgaaaaggt cgactcgaag aatatcttct
      961 tactgtacga ttctatggat gacgccttgc gtgctgatga tatcaagatc caccttacat
     1021 tctctgccga ccatgggtta caacccgacg ctgagattcg tttcaatgga attaagattg
     1081 gtcacatcac tgatattgaa atgaagagcg atctgcgtac agtgaatgca acagcttaca
     1141 ttgataagag tatggaagct ttattaggcg cggacgcata cttttggacc gtcaatgcgc
     1201 gtttcaacgc tgaagggatc agcaacttag atactttaat caagggggct tatgtaaaca
     1261 ttttgcctgg gacagggaaa agcgcacgca cgttcgaagt acacaattcg cgcccggtga
     1321 acatcacaga taatacgggt cttaatttag tgctggagac tgaccgctta ggttctttag
     1381 gttacaataa acccgtttac tatcgtcagg tccaagtagg gcacactact ggctacgagt
     1441 tgagtccgac cgggcagaat gttttaatct atgtgaacat ccacgagccg tatgtgaacc
     1501 ttgtacgtga gaatacgaaa ttttggaaca gtaccggttt tcgtatcaaa ggaggattga
     1561 tgaccgaaat gcgcatcagt accgaatccg ttgccgcgat catcggagga ggaatttcat
     1621 tttctacgcc ggataaagac gacatgggaa accgtgtagc aaacggaaag cactttgttc
     1681 ttcatcgtga tccaaacgat aagtggctga cgtggtcacc gccattgtcc cttggcaaga
     1741 tcgacaccac tccaccccag gaaaaagaga acaatgaagg cgagacccat caccaccatc
     1801 accattaagg ccaggccggc cgatccggct gctaacaaag cccgaaagga agctgagttg
     1861 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     1921 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
     1981 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2041 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     2101 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     2161 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     2221 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     2281 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     2341 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     2401 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaacgacga cctgcagcca
     2461 agctagcttg gctggacgta aactcctctt cagacctaat aacttcgtat agcatacatt
     2521 atacgaagtt atattaaggg ttattgaata tgatcggaat tgggctgcag gaattcgata
     2581 gcttggctgc aggtcgacct cgaggatgtg cgcggaaccc ctatttgttt atttttctaa
     2641 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     2701 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     2761 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     2821 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     2881 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     2941 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     3001 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     3061 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     3121 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     3181 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     3241 cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa
     3301 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     3361 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     3421 ggtgagcgtg gctctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     3481 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     3541 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     3601 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     3661 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3721 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3781 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     3841 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     3901 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     3961 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     4021 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     4081 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     4141 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     4201 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     4261 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     4321 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     4381 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     4441 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     4501 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     4561 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     4621 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     4681 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     4741 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     4801 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     4861 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     4921 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     4981 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     5041 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     5101 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     5161 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     5221 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     5281 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     5341 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     5401 cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa
     5461 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
     5521 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
     5581 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
     5641 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     5701 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     5761 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5821 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     5881 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     5941 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     6001 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca
     6061 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     6121 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     6181 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     6241 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     6301 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     6361 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     6421 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     6481 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     6541 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     6601 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     6661 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     6721 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     6781 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     6841 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     6901 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     6961 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     7021 tgcaaggaga tggcgcccaa
//
