LOCUS       sequence-309852-        6656 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses MlaFB, His tag on MlaF C-term.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6656)
  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 6656)
  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: pBEL1306
FEATURES             Location/Qualifiers
     source          1..6656
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     26..45
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     129..148
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        129..147
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    148..172
                     /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             1033..1050
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1425..1443)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1439..1486
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1523..1978
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1610..1629)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1820..1841
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(2053..2086)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2165..2269
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2270..2338,2339..3130)
                     /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(2488..2507)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3301..3889
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3790..3809
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4043..4060
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4075..4214
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4140..4159)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4300..4322
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4316..4507)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5316..6398)
                     /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     6360..6379
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6399..6476)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
       61 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc
      121 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
      181 aataattttg tttaactttg gcccaggcgg ccaggagggt ctcacatgga gcagtctgtg
      241 gcgaatttag tcgatatgcg cgatgtcagt tttacgcgtg gcaatcgctg catcttcgat
      301 aatatttccc tgaccgtgcc gcgagggaag atcacggcga tcatggggcc atcgggcatc
      361 ggtaaaacga cgctactccg tctgattggc gggcaaatcg caccagatca tggtgagatc
      421 cttttcgatg gtgagaatat tccggcgatg tctcgttcgc gcctgtatac agtgcgcaaa
      481 cggatgagca tgttatttca gtccggggcg ttgttcactg atatgaacgt atttgacaac
      541 gtcgcctatc cactgcgcga acatacccaa cttcccgcgc cattgttgca tagtacggtg
      601 atgatgaagc tggaggccgt ggggctgcgt ggagcggcta aactaatgcc ttctgaactt
      661 tccggtggga tggcgcggcg tgcagcgctg gcgcgtgcga ttgcgctgga gccggatctc
      721 atcatgtttg atgaaccttt tgttgggcaa gatcccatca ccatgggcgt actggtgaag
      781 ctgatttctg agctgaacag cgcgctgggc gtgacttgtg tggtggtttc tcacgatgtg
      841 ccggaagtgt taagtattgc ggatcacgcc tggatcctgg cggacaaaaa aattgtcgct
      901 catggcagtg cccaggcgtt gcaggcgaat cctgatccgc gcgtacgtca gtttctggac
      961 gggatagctg acgggcctgt tccgttccgc tatcctgccg gcgattatca cgctgatctt
     1021 ttaccaggga gtcatcacca ccatcaccat taagccactc atgagcgagt cactgagctg
     1081 gatgcagacg ggtgacacgc tggcgttatc cggagagctg gatcaggacg ttttgctacc
     1141 gctttgggaa atgcgtgagg aagcggtgaa ggggattacc tgcatcgatc ttagccgtgt
     1201 ctcccgcgtg gatacggggg gactggcact gctgctccat cttatcgatc tggcgaaaaa
     1261 gcagggcaac aatgtgacgc ttcagggggt aaacgacaaa gtgtataccc tggcgaaatt
     1321 gtataatttg cctgctgatg ttctgcctcg ttaagagaga ccggccaggc cggccgatcc
     1381 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     1441 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     1501 tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     1561 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     1621 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     1681 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     1741 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     1801 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     1861 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     1921 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     1981 gtggcacttt tcggggaacg acgacctgca gccaagctag cttggctgga cgtaaactcc
     2041 tcttcagacc taataacttc gtatagcata cattatacga agttatatta agggttattg
     2101 aatatgatcg gaattgggct gcaggaattc gatagcttgg ctgcaggtcg acctcgagga
     2161 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2221 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     2281 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     2341 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     2401 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     2461 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     2521 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     2581 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     2641 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     2701 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     2761 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat
     2821 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     2881 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     2941 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggctctc gcggtatcat
     3001 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     3061 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     3121 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     3181 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     3241 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3301 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     3361 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     3421 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     3481 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     3541 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     3601 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     3661 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     3721 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     3781 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3841 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3901 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     3961 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     4021 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     4081 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
     4141 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     4201 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     4261 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     4321 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     4381 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     4441 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     4501 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     4561 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     4621 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     4681 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     4741 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     4801 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     4861 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     4921 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     4981 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     5041 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     5101 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     5161 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     5221 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     5281 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5341 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5401 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     5461 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     5521 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     5581 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     5641 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     5701 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     5761 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     5821 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     5881 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     5941 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     6001 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     6061 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     6121 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     6181 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     6241 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     6301 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     6361 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     6421 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     6481 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     6541 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     6601 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccga
//
