LOCUS       sequence-309897-        6698 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses (Strep-MlaF_E170Q)-(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: pBEL2077
FEATURES             Location/Qualifiers
     source          1..6698
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     563..582
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(602..679)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     885..904
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     988..1007
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        988..1006
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1007..1031
                     /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             1088..1111
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1929..1946
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1947..1967
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(2326..2344)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2340..2387
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2424..2879
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2511..2530)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2721..2742
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(2954..2987)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3066..3170
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3171..3239,3240..4031)
                     /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(3389..3408)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4202..4790
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4691..4710
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4944..4961
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4976..5115
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5041..5060)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5201..5223
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5217..5408)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6217..6698,1..601))
                     /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"
ORIGIN
        1 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
       61 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
      121 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
      181 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
      241 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
      301 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
      361 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
      421 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
      481 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
      541 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
      601 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
      661 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
      721 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
      781 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
      841 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
      901 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
      961 aggatcgaga tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg
     1021 ataacaattc ccctctagaa ataattttgt ttaactttgg cccaggcggc caggagggtc
     1081 tcacatgtgg agccacccgc agttcgaaaa agagcagtct gtggcgaatt tagtcgatat
     1141 gcgcgatgtc agttttacgc gtggcaatcg ctgcatcttc gataatattt ccctgaccgt
     1201 gccgcgaggg aagatcacgg cgatcatggg gccatcgggc atcggtaaaa cgacgctact
     1261 ccgtctgatt ggcgggcaaa tcgcaccaga tcatggtgag atccttttcg atggtgagaa
     1321 tattccggcg atgtctcgtt cgcgcctgta tacagtgcgc aaacggatga gcatgttatt
     1381 tcagtccggg gcgttgttca ctgatatgaa cgtatttgac aacgtcgcct atccactgcg
     1441 cgaacatacc caacttcccg cgccattgtt gcatagtacg gtgatgatga agctggaggc
     1501 cgtggggctg cgtggagcgg ctaaactaat gccttctgaa ctttccggtg ggatggcgcg
     1561 gcgtgcagcg ctggcgcgtg cgattgcgct ggagccggat ctcatcatgt ttgatcagcc
     1621 ttttgttggg caagatccca tcaccatggg cgtactggtg aagctgattt ctgagctgaa
     1681 cagcgcgctg ggcgtgactt gtgtggtggt ttctcacgat gtgccggaag tgttaagtat
     1741 tgcggatcac gcctggatcc tggcggacaa aaaaattgtc gctcatggca gtgcccaggc
     1801 gttgcaggcg aatcctgatc cgcgcgtacg tcagtttctg gacgggatag ctgacgggcc
     1861 tgttccgttc cgctatcctg ccggcgatta tcacgctgat cttttaccag ggagttaagc
     1921 cactcatgca tcaccaccat caccatgaaa atctgtattt tcagagcgag tcactgagct
     1981 ggatgcagac gggtgacacg ctggcgttat ccggagagct ggatcaggac gttttgctac
     2041 cgctttggga aatgcgtgag gaagcggtga aggggattac ctgcatcgat cttagccgtg
     2101 tctcccgcgt ggatacgggg ggactggcac tgctgctcca tcttatcgat ctggcgaaaa
     2161 agcagggcaa caatgtgacg cttcaggggg taaacgacaa agtgtatacc ctggcgaaat
     2221 tgtataattt gcctgctgat gttctgcctc gttaagagag accggccagg ccggccgatc
     2281 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
     2341 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     2401 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2461 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     2521 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2581 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     2641 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     2701 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2761 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     2821 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     2881 ggtggcactt ttcggggaac gacgacctgc agccaagcta gcttggctgg acgtaaactc
     2941 ctcttcagac ctaataactt cgtatagcat acattatacg aagttatatt aagggttatt
     3001 gaatatgatc ggaattgggc tgcaggaatt cgatagcttg gctgcaggtc gacctcgagg
     3061 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     3121 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     3181 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     3241 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     3301 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     3361 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     3421 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     3481 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     3541 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     3601 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     3661 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa
     3721 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     3781 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     3841 cggctggctg gtttattgct gataaatctg gagccggtga gcgtggctct cgcggtatca
     3901 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     3961 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     4021 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     4081 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     4141 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4201 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4261 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4321 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     4381 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     4441 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     4501 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     4561 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     4621 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     4681 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     4741 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     4801 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     4861 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     4921 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     4981 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
     5041 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     5101 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     5161 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     5221 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     5281 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     5341 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     5401 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     5461 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     5521 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     5581 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     5641 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     5701 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     5761 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     5821 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc
     5881 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
     5941 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
     6001 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
     6061 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
     6121 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     6181 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     6241 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     6301 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     6361 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     6421 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     6481 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     6541 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     6601 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     6661 tatcggctga atttgattgc gagtgagata tttatgcc
//
