LOCUS       sequence-309901-        6632 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses (Strep-MlaF_Delta[248-269])-(6xHis-TEV-MlaB).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6632)
  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 6632)
  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: pBEL2082
FEATURES             Location/Qualifiers
     source          1..6632
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     42..61
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     145..164
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        145..163
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    164..188
                     /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             245..268
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1020..1037
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1038..1058
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(1417..1435)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1431..1478
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1515..1970
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1602..1621)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1812..1833
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(2045..2078)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2157..2261
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2262..2330,2331..3122)
                     /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(2480..2499)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3293..3881
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3782..3801
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4035..4052
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4067..4206
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4132..4151)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4292..4314
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4308..4499)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5308..6390)
                     /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     6352..6371
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6391..6468)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
       61 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
      121 atcgagatct cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata
      181 acaattcccc tctagaaata attttgttta actttggccc aggcggccag gagggtctca
      241 catgtggagc cacccgcagt tcgaaaaaga gcagtctgtg gcgaatttag tcgatatgcg
      301 cgatgtcagt tttacgcgtg gcaatcgctg catcttcgat aatatttccc tgaccgtgcc
      361 gcgagggaag atcacggcga tcatggggcc atcgggcatc ggtaaaacga cgctactccg
      421 tctgattggc gggcaaatcg caccagatca tggtgagatc cttttcgatg gtgagaatat
      481 tccggcgatg tctcgttcgc gcctgtatac agtgcgcaaa cggatgagca tgttatttca
      541 gtccggggcg ttgttcactg atatgaacgt atttgacaac gtcgcctatc cactgcgcga
      601 acatacccaa cttcccgcgc cattgttgca tagtacggtg atgatgaagc tggaggccgt
      661 ggggctgcgt ggagcggcta aactaatgcc ttctgaactt tccggtggga tggcgcggcg
      721 tgcagcgctg gcgcgtgcga ttgcgctgga gccggatctc atcatgtttg atgaaccttt
      781 tgttgggcaa gatcccatca ccatgggcgt actggtgaag ctgatttctg agctgaacag
      841 cgcgctgggc gtgacttgtg tggtggtttc tcacgatgtg ccggaagtgt taagtattgc
      901 ggatcacgcc tggatcctgg cggacaaaaa aattgtcgct catggcagtg cccaggcgtt
      961 gcaggcgaat cctgatccgc gcgtacgtca gtttctggac gggatataag ccactcatgc
     1021 atcaccacca tcaccatgaa aatctgtatt ttcagagcga gtcactgagc tggatgcaga
     1081 cgggtgacac gctggcgtta tccggagagc tggatcagga cgttttgcta ccgctttggg
     1141 aaatgcgtga ggaagcggtg aaggggatta cctgcatcga tcttagccgt gtctcccgcg
     1201 tggatacggg gggactggca ctgctgctcc atcttatcga tctggcgaaa aagcagggca
     1261 acaatgtgac gcttcagggg gtaaacgaca aagtgtatac cctggcgaaa ttgtataatt
     1321 tgcctgctga tgttctgcct cgttaagaga gaccggccag gccggccgat ccggctgcta
     1381 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     1441 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     1501 gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     1561 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     1621 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     1681 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     1741 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     1801 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     1861 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     1921 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     1981 tttcggggaa cgacgacctg cagccaagct agcttggctg gacgtaaact cctcttcaga
     2041 cctaataact tcgtatagca tacattatac gaagttatat taagggttat tgaatatgat
     2101 cggaattggg ctgcaggaat tcgatagctt ggctgcaggt cgacctcgag gatgtgcgcg
     2161 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     2221 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     2281 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     2341 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     2401 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     2461 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     2521 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     2581 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     2641 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     2701 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     2761 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa
     2821 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     2881 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     2941 ggtttattgc tgataaatct ggagccggtg agcgtggctc tcgcggtatc attgcagcac
     3001 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     3061 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     3121 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     3181 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     3241 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     3301 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     3361 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     3421 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     3481 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     3541 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     3601 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3661 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     3721 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     3781 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3841 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3901 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3961 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     4021 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     4081 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     4141 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     4201 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     4261 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     4321 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     4381 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     4441 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     4501 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     4561 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     4621 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     4681 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     4741 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     4801 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     4861 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     4921 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg
     4981 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
     5041 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
     5101 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
     5161 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
     5221 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     5281 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     5341 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5401 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     5461 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     5521 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     5581 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     5641 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     5701 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     5761 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     5821 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     5881 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     5941 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     6001 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     6061 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     6121 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     6181 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     6241 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     6301 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     6361 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     6421 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     6481 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     6541 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     6601 cggggcctgc caccataccc acgccgaaac aa
//
