LOCUS       sequence-309858-        6660 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses MlaFB, His tag on MlaB C-term.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6660)
  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 6660)
  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: pBEL1308
FEATURES             Location/Qualifiers
     source          1..6660
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     32..51
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     135..154
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        135..153
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    154..178
                     /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             1352..1369
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1435..1453)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1449..1496
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1533..1988
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1620..1639)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1830..1851
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(2063..2096)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2175..2279
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2280..2348,2349..3140)
                     /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(2498..2517)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3311..3899
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3800..3819
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4053..4070
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4085..4224
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4150..4169)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4310..4332
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4326..4517)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5326..6408)
                     /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     6370..6389
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6409..6486)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
       61 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
      121 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
      181 tctagaaata attttgttta actttggccc aggcggccag gagggtctca catggagcag
      241 tctgtggcga atttagtcga tatgcgcgat gtcagtttta cgcgtggcaa tcgctgcatc
      301 ttcgataata tttccctgac cgtgccgcga gggaagatca cggcgatcat ggggccatcg
      361 ggcatcggta aaacgacgct actccgtctg attggcgggc aaatcgcacc agatcatggt
      421 gagatccttt tcgatggtga gaatattccg gcgatgtctc gttcgcgcct gtatacagtg
      481 cgcaaacgga tgagcatgtt atttcagtcc ggggcgttgt tcactgatat gaacgtattt
      541 gacaacgtcg cctatccact gcgcgaacat acccaacttc ccgcgccatt gttgcatagt
      601 acggtgatga tgaagctgga ggccgtgggg ctgcgtggag cggctaaact aatgccttct
      661 gaactttccg gtgggatggc gcggcgtgca gcgctggcgc gtgcgattgc gctggagccg
      721 gatctcatca tgtttgatga accttttgtt gggcaagatc ccatcaccat gggcgtactg
      781 gtgaagctga tttctgagct gaacagcgcg ctgggcgtga cttgtgtggt ggtttctcac
      841 gatgtgccgg aagtgttaag tattgcggat cacgcctgga tcctggcgga caaaaaaatt
      901 gtcgctcatg gcagtgccca ggcgttgcag gcgaatcctg atccgcgcgt acgtcagttt
      961 ctggacggga tagctgacgg gcctgttccg ttccgctatc ctgccggcga ttatcacgct
     1021 gatcttttac cagggagtta agccactcat gagcgagtca ctgagctgga tgcagacggg
     1081 tgacacgctg gcgttatccg gagagctgga tcaggacgtt ttgctaccgc tttgggaaat
     1141 gcgtgaggaa gcggtgaagg ggattacctg catcgatctt agccgtgtct cccgcgtgga
     1201 tacgggggga ctggcactgc tgctccatct tatcgatctg gcgaaaaagc agggcaacaa
     1261 tgtgacgctt cagggggtaa acgacaaagt gtataccctg gcgaaattgt ataatttgcc
     1321 tgctgatgtt ctgcctcgtt cgggcgagac ccatcaccac catcaccatt aaggccaggc
     1381 cggccgatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     1441 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     1501 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     1561 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     1621 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     1681 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     1741 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     1801 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     1861 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     1921 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     1981 tacaatttag gtggcacttt tcggggaacg acgacctgca gccaagctag cttggctgga
     2041 cgtaaactcc tcttcagacc taataacttc gtatagcata cattatacga agttatatta
     2101 agggttattg aatatgatcg gaattgggct gcaggaattc gatagcttgg ctgcaggtcg
     2161 acctcgagga tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     2221 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     2281 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     2341 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     2401 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     2461 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     2521 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     2581 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     2641 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     2701 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     2761 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     2821 ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     2881 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     2941 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggctctc
     3001 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     3061 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     3121 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     3181 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     3241 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     3301 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     3361 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     3421 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     3481 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     3541 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     3601 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     3661 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     3721 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     3781 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     3841 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     3901 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     3961 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     4021 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     4081 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
     4141 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     4201 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     4261 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     4321 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     4381 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     4441 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     4501 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     4561 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     4621 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     4681 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     4741 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     4801 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     4861 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     4921 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     4981 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
     5041 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     5101 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
     5161 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     5221 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     5281 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     5341 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     5401 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     5461 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     5521 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     5581 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     5641 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     5701 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     5761 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     5821 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     5881 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     5941 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     6001 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     6061 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     6121 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     6181 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     6241 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     6301 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     6361 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     6421 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     6481 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     6541 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     6601 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
//
