LOCUS       sequence-310633-        6698 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Strep-MlaF_L266A)-(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: pBEL2080
FEATURES             Location/Qualifiers
     source          1..6698
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     945..964
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(984..1061)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1267..1286
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1370..1389
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1370..1388
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1389..1413
                     /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             1470..1493
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2311..2328
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2329..2349
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(2708..2726)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2722..2769
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2806..3261
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2893..2912)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3103..3124
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(3336..3369)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3448..3552
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3553..3621,3622..4413)
                     /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(3771..3790)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4584..5172
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5073..5092
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5326..5343
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5358..5497
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5423..5442)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5583..5605
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5599..5790)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6599..6698,1..983))
                     /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 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
       61 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
      121 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
      181 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
      241 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
      301 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
      361 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
      421 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
      481 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
      541 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
      601 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
      661 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
      721 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
      781 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
      841 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
      901 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
      961 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     1021 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     1081 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     1141 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     1201 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     1261 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     1321 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc
     1381 actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt
     1441 ggcccaggcg gccaggaggg tctcacatgt ggagccaccc gcagttcgaa aaagagcagt
     1501 ctgtggcgaa tttagtcgat atgcgcgatg tcagttttac gcgtggcaat cgctgcatct
     1561 tcgataatat ttccctgacc gtgccgcgag ggaagatcac ggcgatcatg gggccatcgg
     1621 gcatcggtaa aacgacgcta ctccgtctga ttggcgggca aatcgcacca gatcatggtg
     1681 agatcctttt cgatggtgag aatattccgg cgatgtctcg ttcgcgcctg tatacagtgc
     1741 gcaaacggat gagcatgtta tttcagtccg gggcgttgtt cactgatatg aacgtatttg
     1801 acaacgtcgc ctatccactg cgcgaacata cccaacttcc cgcgccattg ttgcatagta
     1861 cggtgatgat gaagctggag gccgtggggc tgcgtggagc ggctaaacta atgccttctg
     1921 aactttccgg tgggatggcg cggcgtgcag cgctggcgcg tgcgattgcg ctggagccgg
     1981 atctcatcat gtttgatgaa ccttttgttg ggcaagatcc catcaccatg ggcgtactgg
     2041 tgaagctgat ttctgagctg aacagcgcgc tgggcgtgac ttgtgtggtg gtttctcacg
     2101 atgtgccgga agtgttaagt attgcggatc acgcctggat cctggcggac aaaaaaattg
     2161 tcgctcatgg cagtgcccag gcgttgcagg cgaatcctga tccgcgcgta cgtcagtttc
     2221 tggacgggat agctgacggg cctgttccgt tccgctatcc tgccggcgat tatcacgctg
     2281 atcttgcgcc agggagttaa gccactcatg catcaccacc atcaccatga aaatctgtat
     2341 tttcagagcg agtcactgag ctggatgcag acgggtgaca cgctggcgtt atccggagag
     2401 ctggatcagg acgttttgct accgctttgg gaaatgcgtg aggaagcggt gaaggggatt
     2461 acctgcatcg atcttagccg tgtctcccgc gtggatacgg ggggactggc actgctgctc
     2521 catcttatcg atctggcgaa aaagcagggc aacaatgtga cgcttcaggg ggtaaacgac
     2581 aaagtgtata ccctggcgaa attgtataat ttgcctgctg atgttctgcc tcgttaagag
     2641 agaccggcca ggccggccga tccggctgct aacaaagccc gaaaggaagc tgagttggct
     2701 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     2761 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
     2821 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     2881 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     2941 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     3001 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     3061 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     3121 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     3181 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     3241 aaatattaac gcttacaatt taggtggcac ttttcgggga acgacgacct gcagccaagc
     3301 tagcttggct ggacgtaaac tcctcttcag acctaataac ttcgtatagc atacattata
     3361 cgaagttata ttaagggtta ttgaatatga tcggaattgg gctgcaggaa ttcgatagct
     3421 tggctgcagg tcgacctcga ggatgtgcgc ggaaccccta tttgtttatt tttctaaata
     3481 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     3541 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     3601 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     3661 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     3721 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     3781 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     3841 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     3901 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     3961 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     4021 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     4081 gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     4141 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     4201 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     4261 gagcgtggct ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     4321 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     4381 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     4441 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     4501 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     4561 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     4621 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     4681 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     4741 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     4801 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     4861 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     4921 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     4981 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     5041 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     5101 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     5161 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     5221 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     5281 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     5341 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     5401 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     5461 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     5521 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     5581 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     5641 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     5701 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     5761 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     5821 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     5881 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     5941 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     6001 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     6061 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     6121 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     6181 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     6241 gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     6301 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     6361 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     6421 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     6481 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     6541 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     6601 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     6661 cgcggggaga ggcggtttgc gtattgggcg ccagggtg
//
