LOCUS       sequence-309896-        6698 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses (Strep-MlaF_K47A)-(6xHis-TEV-MlaB).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6698)
  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 6698)
  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: pBEL2076
FEATURES             Location/Qualifiers
     source          1..6698
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(60..1142)
                     /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     1104..1123
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1143..1220)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1426..1445
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1529..1548
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1529..1547
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1548..1572
                     /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             1629..1652
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2470..2487
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2488..2508
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(2867..2885)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2881..2928
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2965..3420
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3052..3071)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3262..3283
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(3495..3528)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3607..3711
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3712..3780,3781..4572)
                     /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(3930..3949)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4743..5331
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5232..5251
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5485..5502
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5517..5656
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5582..5601)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5742..5764
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5758..5949)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
       61 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
      121 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
      181 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
      241 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
      301 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc
      361 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
      421 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
      481 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
      541 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
      601 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
      661 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
      721 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
      781 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
      841 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
      901 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
      961 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     1021 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     1081 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     1141 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     1201 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
     1261 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     1321 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     1381 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     1441 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
     1501 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
     1561 gataacaatt cccctctaga aataattttg tttaactttg gcccaggcgg ccaggagggt
     1621 ctcacatgtg gagccacccg cagttcgaaa aagagcagtc tgtggcgaat ttagtcgata
     1681 tgcgcgatgt cagttttacg cgtggcaatc gctgcatctt cgataatatt tccctgaccg
     1741 tgccgcgagg gaagatcacg gcgatcatgg ggccatcggg catcggtgcg acgacgctac
     1801 tccgtctgat tggcgggcaa atcgcaccag atcatggtga gatccttttc gatggtgaga
     1861 atattccggc gatgtctcgt tcgcgcctgt atacagtgcg caaacggatg agcatgttat
     1921 ttcagtccgg ggcgttgttc actgatatga acgtatttga caacgtcgcc tatccactgc
     1981 gcgaacatac ccaacttccc gcgccattgt tgcatagtac ggtgatgatg aagctggagg
     2041 ccgtggggct gcgtggagcg gctaaactaa tgccttctga actttccggt gggatggcgc
     2101 ggcgtgcagc gctggcgcgt gcgattgcgc tggagccgga tctcatcatg tttgatgaac
     2161 cttttgttgg gcaagatccc atcaccatgg gcgtactggt gaagctgatt tctgagctga
     2221 acagcgcgct gggcgtgact tgtgtggtgg tttctcacga tgtgccggaa gtgttaagta
     2281 ttgcggatca cgcctggatc ctggcggaca aaaaaattgt cgctcatggc agtgcccagg
     2341 cgttgcaggc gaatcctgat ccgcgcgtac gtcagtttct ggacgggata gctgacgggc
     2401 ctgttccgtt ccgctatcct gccggcgatt atcacgctga tcttttacca gggagttaag
     2461 ccactcatgc atcaccacca tcaccatgaa aatctgtatt ttcagagcga gtcactgagc
     2521 tggatgcaga cgggtgacac gctggcgtta tccggagagc tggatcagga cgttttgcta
     2581 ccgctttggg aaatgcgtga ggaagcggtg aaggggatta cctgcatcga tcttagccgt
     2641 gtctcccgcg tggatacggg gggactggca ctgctgctcc atcttatcga tctggcgaaa
     2701 aagcagggca acaatgtgac gcttcagggg gtaaacgaca aagtgtatac cctggcgaaa
     2761 ttgtataatt tgcctgctga tgttctgcct cgttaagaga gaccggccag gccggccgat
     2821 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     2881 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     2941 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     3001 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     3061 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     3121 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     3181 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     3241 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     3301 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     3361 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     3421 aggtggcact tttcggggaa cgacgacctg cagccaagct agcttggctg gacgtaaact
     3481 cctcttcaga cctaataact tcgtatagca tacattatac gaagttatat taagggttat
     3541 tgaatatgat cggaattggg ctgcaggaat tcgatagctt ggctgcaggt cgacctcgag
     3601 gatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     3661 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     3721 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     3781 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     3841 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     3901 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     3961 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     4021 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     4081 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     4141 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     4201 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca
     4261 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     4321 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     4381 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggctc tcgcggtatc
     4441 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     4501 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     4561 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     4621 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     4681 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     4741 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     4801 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     4861 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     4921 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     4981 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     5041 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     5101 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     5161 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     5221 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     5281 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     5341 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     5401 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     5461 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     5521 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca
     5581 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     5641 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     5701 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     5761 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
     5821 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc
     5881 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
     5941 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
     6001 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga
     6061 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca
     6121 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca
     6181 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact
     6241 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
     6301 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
     6361 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc
     6421 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     6481 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     6541 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     6601 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     6661 ccaccggaag gagctgactg ggttgaaggc tctcaagg
//
