LOCUS       sequence-309853-        6674 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses MlaFB, His tag on MlaB N-term.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6674)
  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 6674)
  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: pBEL1307
FEATURES             Location/Qualifiers
     source          1..6674
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     30..49
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     133..152
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        133..151
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    152..176
                     /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             1050..1067
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1068..1088
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(1447..1465)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1461..1508
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1545..2000
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1632..1651)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1842..1863
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(2075..2108)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2187..2291
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2292..2360,2361..3152)
                     /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(2510..2529)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3323..3911
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3812..3831
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4065..4082
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4097..4236
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4162..4181)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4322..4344
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4338..4529)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5338..6420)
                     /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     6382..6401
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6421..6498)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
       61 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg
      121 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctc
      181 tagaaataat tttgtttaac tttggcccag gcggccagga gggtctcaca tggagcagtc
      241 tgtggcgaat ttagtcgata tgcgcgatgt cagttttacg cgtggcaatc gctgcatctt
      301 cgataatatt tccctgaccg tgccgcgagg gaagatcacg gcgatcatgg ggccatcggg
      361 catcggtaaa acgacgctac tccgtctgat tggcgggcaa atcgcaccag atcatggtga
      421 gatccttttc gatggtgaga atattccggc gatgtctcgt tcgcgcctgt atacagtgcg
      481 caaacggatg agcatgttat ttcagtccgg ggcgttgttc actgatatga acgtatttga
      541 caacgtcgcc tatccactgc gcgaacatac ccaacttccc gcgccattgt tgcatagtac
      601 ggtgatgatg aagctggagg ccgtggggct gcgtggagcg gctaaactaa tgccttctga
      661 actttccggt gggatggcgc ggcgtgcagc gctggcgcgt gcgattgcgc tggagccgga
      721 tctcatcatg tttgatgaac cttttgttgg gcaagatccc atcaccatgg gcgtactggt
      781 gaagctgatt tctgagctga acagcgcgct gggcgtgact tgtgtggtgg tttctcacga
      841 tgtgccggaa gtgttaagta ttgcggatca cgcctggatc ctggcggaca aaaaaattgt
      901 cgctcatggc agtgcccagg cgttgcaggc gaatcctgat ccgcgcgtac gtcagtttct
      961 ggacgggata gctgacgggc ctgttccgtt ccgctatcct gccggcgatt atcacgctga
     1021 tcttttacca gggagttaag ccactcatgc atcaccacca tcaccatgaa aatctgtatt
     1081 ttcagagcga gtcactgagc tggatgcaga cgggtgacac gctggcgtta tccggagagc
     1141 tggatcagga cgttttgcta ccgctttggg aaatgcgtga ggaagcggtg aaggggatta
     1201 cctgcatcga tcttagccgt gtctcccgcg tggatacggg gggactggca ctgctgctcc
     1261 atcttatcga tctggcgaaa aagcagggca acaatgtgac gcttcagggg gtaaacgaca
     1321 aagtgtatac cctggcgaaa ttgtataatt tgcctgctga tgttctgcct cgttaagaga
     1381 gaccggccag gccggccgat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     1441 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     1501 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     1561 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     1621 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     1681 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     1741 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     1801 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     1861 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     1921 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     1981 aatattaacg cttacaattt aggtggcact tttcggggaa cgacgacctg cagccaagct
     2041 agcttggctg gacgtaaact cctcttcaga cctaataact tcgtatagca tacattatac
     2101 gaagttatat taagggttat tgaatatgat cggaattggg ctgcaggaat tcgatagctt
     2161 ggctgcaggt cgacctcgag gatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     2221 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     2281 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     2341 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     2401 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     2461 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     2521 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     2581 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     2641 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     2701 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     2761 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     2821 acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     2881 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     2941 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     3001 agcgtggctc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     3061 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     3121 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     3181 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     3241 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     3301 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     3361 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     3421 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     3481 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     3541 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     3601 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     3661 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     3721 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     3781 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     3841 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     3901 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     3961 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     4021 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     4081 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     4141 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     4201 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     4261 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     4321 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     4381 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     4441 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     4501 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     4561 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     4621 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     4681 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     4741 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     4801 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     4861 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     4921 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     4981 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
     5041 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
     5101 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
     5161 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
     5221 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     5281 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     5341 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     5401 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     5461 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     5521 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     5581 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     5641 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     5701 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     5761 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     5821 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     5881 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     5941 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     6001 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     6061 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     6121 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     6181 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     6241 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     6301 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     6361 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     6421 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     6481 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     6541 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     6601 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     6661 aagcgctcat gagc
//
