LOCUS       sequence-309841-        5841 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses MlaB in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5841)
  AUTHORS   Kolich LR, Chang YT, Coudray N, Giacometti SI, MacRae MR, Isom GL, 
            Teran EM, Bhabha G, Ekiert DC
  TITLE     Structure of MlaFB uncovers novel mechanisms of ABC transporter 
            regulation.
  JOURNAL   Elife. 2020 Jun 30;9. pii: 60030. doi: 10.7554/eLife.60030.
  PUBMED    32602838
REFERENCE   2  (bases 1 to 5841)
  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"; journalName: "Elife."; 
            date: "2020-06-30"; volume: "9. pii: 60030. doi"; pages: " 
            10.7554/eLife.60030"
COMMENT     Sequence Label: pBEL1246
FEATURES             Location/Qualifiers
     source          1..5841
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     59..78
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     162..181
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        162..180
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    181..205
                     /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             254..271
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             272..292
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(643..661)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      657..704
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      741..1196
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(828..847)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1038..1059
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(1271..1304)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1383..1487
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1488..1556,1557..2348)
                     /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(1706..1725)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2519..3107
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3008..3027
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3261..3278
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3293..3432
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3358..3377)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3518..3540
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3534..3725)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4534..5616)
                     /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     5578..5597
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5617..5694)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
       61 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
      121 tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg
      181 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttggcccagg
      241 agatatacat atgcatcacc accatcacca tgaaaatctg tattttcaga gcgagtcact
      301 gagctggatg cagacgggtg acacgctggc gttatccgga gagctggatc aggacgtttt
      361 gctaccgctt tgggaaatgc gtgaggaagc ggtgaagggg attacctgca tcgatcttag
      421 ccgtgtctcc cgcgtggata cggggggact ggcactgctg ctccatctta tcgatctggc
      481 gaaaaagcag ggcaacaatg tgacgcttca gggggtaaac gacaaagtgt ataccctggc
      541 gaaattgtat aatttgcctg ctgatgttct gcctcgttaa agaccggccg gccgatccgg
      601 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag
      661 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
      721 tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
      781 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
      841 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
      901 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
      961 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     1021 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     1081 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     1141 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt
     1201 ggcacttttc ggggaacgac gacctgcagc caagctagct tggctggacg taaactcctc
     1261 ttcagaccta ataacttcgt atagcataca ttatacgaag ttatattaag ggttattgaa
     1321 tatgatcgga attgggctgc aggaattcga tagcttggct gcaggtcgac ctcgaggatg
     1381 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     1441 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     1501 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     1561 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     1621 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     1681 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     1741 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     1801 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     1861 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     1921 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     1981 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg
     2041 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     2101 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     2161 ctggctggtt tattgctgat aaatctggag ccggtgagcg tggctctcgc ggtatcattg
     2221 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     2281 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     2341 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     2401 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     2461 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     2521 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     2581 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     2641 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     2701 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     2761 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     2821 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     2881 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     2941 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     3001 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     3061 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     3121 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     3181 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     3241 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     3301 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac
     3361 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     3421 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     3481 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     3541 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     3601 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     3661 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     3721 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     3781 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     3841 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     3901 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     3961 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     4021 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     4081 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     4141 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc
     4201 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
     4261 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
     4321 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
     4381 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
     4441 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     4501 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4561 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4621 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     4681 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     4741 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     4801 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     4861 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     4921 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     4981 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     5041 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     5101 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     5161 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     5221 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     5281 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     5341 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     5401 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     5461 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     5521 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     5581 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     5641 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     5701 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     5761 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     5821 cggccacggg gcctgccacc a
//
