LOCUS       sequence-309840-        6363 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses MlaF in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6363)
  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 6363)
  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: pBEL1245
FEATURES             Location/Qualifiers
     source          1..6363
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     969..988
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1008..1085)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1291..1310
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1394..1413
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1394..1412
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1413..1437
                     /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             1486..1503
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1504..1524
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQM"
     primer_bind     complement(2397..2415)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2411..2458
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2495..2950
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2582..2601)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2792..2813
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(3025..3058)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3137..3241
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3242..3310,3311..4102)
                     /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(3460..3479)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4273..4861
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4762..4781
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5015..5032
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5047..5186
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5112..5131)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5272..5294
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5288..5479)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6288..6363,1..1007))
                     /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"
ORIGIN
        1 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
       61 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
      121 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
      181 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
      241 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
      301 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
      361 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
      421 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
      481 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
      541 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
      601 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
      661 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
      721 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
      781 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
      841 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
      901 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
      961 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     1021 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     1081 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     1141 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     1201 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     1261 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
     1321 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc
     1381 gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa caattcccct
     1441 ctagaaataa ttttgtttaa ctttggccca ggagatatac atatgcatca ccaccatcac
     1501 catgaaaatc tgtattttca gatggagcag tctgtggcga atttagtcga tatgcgcgat
     1561 gtcagtttta cgcgtggcaa tcgctgcatc ttcgataata tttccctgac cgtgccgcga
     1621 gggaagatca cggcgatcat ggggccatcg ggcatcggta aaacgacgct actccgtctg
     1681 attggcgggc aaatcgcacc agatcatggt gagatccttt tcgatggtga gaatattccg
     1741 gcgatgtctc gttcgcgcct gtatacagtg cgcaaacgga tgagcatgtt atttcagtcc
     1801 ggggcgttgt tcactgatat gaacgtattt gacaacgtcg cctatccact gcgcgaacat
     1861 acccaacttc ccgcgccatt gttgcatagt acggtgatga tgaagctgga ggccgtgggg
     1921 ctgcgtggag cggctaaact aatgccttct gaactttccg gtgggatggc gcggcgtgca
     1981 gcgctggcgc gtgcgattgc gctggagccg gatctcatca tgtttgatga accttttgtt
     2041 gggcaagatc ccatcaccat gggcgtactg gtgaagctga tttctgagct gaacagcgcg
     2101 ctgggcgtga cttgtgtggt ggtttctcac gatgtgccgg aagtgttaag tattgcggat
     2161 cacgcctgga tcctggcgga caaaaaaatt gtcgctcatg gcagtgccca ggcgttgcag
     2221 gcgaatcctg atccgcgcgt acgtcagttt ctggacggga tagctgacgg gcctgttccg
     2281 ttccgctatc ctgccggcga ttatcacgct gatcttttac cagggagtta agagagaccg
     2341 gccggccgat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     2401 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     2461 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     2521 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     2581 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     2641 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     2701 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     2761 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     2821 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     2881 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     2941 cttacaattt aggtggcact tttcggggaa cgacgacctg cagccaagct agcttggctg
     3001 gacgtaaact cctcttcaga cctaataact tcgtatagca tacattatac gaagttatat
     3061 taagggttat tgaatatgat cggaattggg ctgcaggaat tcgatagctt ggctgcaggt
     3121 cgacctcgag gatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3181 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     3241 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     3301 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     3361 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     3421 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     3481 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     3541 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     3601 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     3661 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     3721 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     3781 gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     3841 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     3901 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggctc
     3961 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     4021 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     4081 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     4141 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     4201 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     4261 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     4321 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     4381 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     4441 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     4501 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     4561 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     4621 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     4681 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     4741 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     4801 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     4861 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     4921 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     4981 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     5041 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     5101 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     5161 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     5221 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     5281 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     5341 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     5401 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     5461 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     5521 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     5581 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     5641 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     5701 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     5761 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     5821 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     5881 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     5941 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     6001 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     6061 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     6121 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     6181 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     6241 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     6301 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     6361 gcg
//
