LOCUS       sequence-309846-        6664 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses MlaFB, His tag on MlaF N-term.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6664)
  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 6664)
  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: pBEL1305
FEATURES             Location/Qualifiers
     source          1..6664
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     58..77
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     161..180
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        161..179
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    180..204
                     /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             253..270
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             271..291
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQM"
     primer_bind     complement(1465..1483)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1479..1526
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1563..2018
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1650..1669)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1860..1881
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(2093..2126)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2205..2309
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2310..2378,2379..3170)
                     /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(2528..2547)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3341..3929
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3830..3849
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4083..4100
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4115..4254
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4180..4199)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4340..4362
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4356..4547)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5356..6438)
                     /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     6400..6419
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6439..6516)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt
       61 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat
      121 gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg
      181 gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt tggcccagga
      241 gatatacata tgcatcacca ccatcaccat gaaaatctgt attttcagat ggagcagtct
      301 gtggcgaatt tagtcgatat gcgcgatgtc agttttacgc gtggcaatcg ctgcatcttc
      361 gataatattt ccctgaccgt gccgcgaggg aagatcacgg cgatcatggg gccatcgggc
      421 atcggtaaaa cgacgctact ccgtctgatt ggcgggcaaa tcgcaccaga tcatggtgag
      481 atccttttcg atggtgagaa tattccggcg atgtctcgtt cgcgcctgta tacagtgcgc
      541 aaacggatga gcatgttatt tcagtccggg gcgttgttca ctgatatgaa cgtatttgac
      601 aacgtcgcct atccactgcg cgaacatacc caacttcccg cgccattgtt gcatagtacg
      661 gtgatgatga agctggaggc cgtggggctg cgtggagcgg ctaaactaat gccttctgaa
      721 ctttccggtg ggatggcgcg gcgtgcagcg ctggcgcgtg cgattgcgct ggagccggat
      781 ctcatcatgt ttgatgaacc ttttgttggg caagatccca tcaccatggg cgtactggtg
      841 aagctgattt ctgagctgaa cagcgcgctg ggcgtgactt gtgtggtggt ttctcacgat
      901 gtgccggaag tgttaagtat tgcggatcac gcctggatcc tggcggacaa aaaaattgtc
      961 gctcatggca gtgcccaggc gttgcaggcg aatcctgatc cgcgcgtacg tcagtttctg
     1021 gacgggatag ctgacgggcc tgttccgttc cgctatcctg ccggcgatta tcacgctgat
     1081 cttttaccag ggagttaagc cactcatgag cgagtcactg agctggatgc agacgggtga
     1141 cacgctggcg ttatccggag agctggatca ggacgttttg ctaccgcttt gggaaatgcg
     1201 tgaggaagcg gtgaagggga ttacctgcat cgatcttagc cgtgtctccc gcgtggatac
     1261 ggggggactg gcactgctgc tccatcttat cgatctggcg aaaaagcagg gcaacaatgt
     1321 gacgcttcag ggggtaaacg acaaagtgta taccctggcg aaattgtata atttgcctgc
     1381 tgatgttctg cctcgttaag agagaccggc cggccgatcc ggctgctaac aaagcccgaa
     1441 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     1501 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
     1561 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     1621 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     1681 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     1741 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     1801 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     1861 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     1921 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     1981 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaacg
     2041 acgacctgca gccaagctag cttggctgga cgtaaactcc tcttcagacc taataacttc
     2101 gtatagcata cattatacga agttatatta agggttattg aatatgatcg gaattgggct
     2161 gcaggaattc gatagcttgg ctgcaggtcg acctcgagga tgtgcgcgga acccctattt
     2221 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     2281 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     2341 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     2401 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     2461 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     2521 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     2581 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     2641 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     2701 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     2761 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     2821 taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac
     2881 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     2941 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     3001 ataaatctgg agccggtgag cgtggctctc gcggtatcat tgcagcactg gggccagatg
     3061 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     3121 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     3181 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     3241 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     3301 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     3361 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     3421 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     3481 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     3541 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     3601 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     3661 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     3721 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     3781 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     3841 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     3901 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     3961 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     4021 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     4081 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     4141 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     4201 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     4261 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     4321 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     4381 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     4441 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     4501 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     4561 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     4621 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     4681 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     4741 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     4801 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     4861 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     4921 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     4981 ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc
     5041 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
     5101 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc
     5161 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag
     5221 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
     5281 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     5341 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     5401 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     5461 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     5521 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     5581 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
     5641 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     5701 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     5761 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     5821 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     5881 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     5941 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     6001 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     6061 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     6121 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     6181 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     6241 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     6301 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     6361 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     6421 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     6481 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     6541 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     6601 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     6661 ccat
//
