LOCUS       sequence-309899-        6698 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses (Strep-MlaF_L265A)-(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: pBEL2079
FEATURES             Location/Qualifiers
     source          1..6698
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     78..97
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     181..200
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        181..199
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    200..224
                     /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             281..304
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1122..1139
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1140..1160
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(1519..1537)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1533..1580
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1617..2072
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1704..1723)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1914..1935
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(2147..2180)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2259..2363
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2364..2432,2433..3224)
                     /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(2582..2601)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3395..3983
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3884..3903
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4137..4154
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4169..4308
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4234..4253)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4394..4416
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4410..4601)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5410..6492)
                     /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     6454..6473
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6493..6570)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
       61 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
      121 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
      181 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
      241 tgtttaactt tggcccaggc ggccaggagg gtctcacatg tggagccacc cgcagttcga
      301 aaaagagcag tctgtggcga atttagtcga tatgcgcgat gtcagtttta cgcgtggcaa
      361 tcgctgcatc ttcgataata tttccctgac cgtgccgcga gggaagatca cggcgatcat
      421 ggggccatcg ggcatcggta aaacgacgct actccgtctg attggcgggc aaatcgcacc
      481 agatcatggt gagatccttt tcgatggtga gaatattccg gcgatgtctc gttcgcgcct
      541 gtatacagtg cgcaaacgga tgagcatgtt atttcagtcc ggggcgttgt tcactgatat
      601 gaacgtattt gacaacgtcg cctatccact gcgcgaacat acccaacttc ccgcgccatt
      661 gttgcatagt acggtgatga tgaagctgga ggccgtgggg ctgcgtggag cggctaaact
      721 aatgccttct gaactttccg gtgggatggc gcggcgtgca gcgctggcgc gtgcgattgc
      781 gctggagccg gatctcatca tgtttgatga accttttgtt gggcaagatc ccatcaccat
      841 gggcgtactg gtgaagctga tttctgagct gaacagcgcg ctgggcgtga cttgtgtggt
      901 ggtttctcac gatgtgccgg aagtgttaag tattgcggat cacgcctgga tcctggcgga
      961 caaaaaaatt gtcgctcatg gcagtgccca ggcgttgcag gcgaatcctg atccgcgcgt
     1021 acgtcagttt ctggacggga tagctgacgg gcctgttccg ttccgctatc ctgccggcga
     1081 ttatcacgct gatgcgttac cagggagtta agccactcat gcatcaccac catcaccatg
     1141 aaaatctgta ttttcagagc gagtcactga gctggatgca gacgggtgac acgctggcgt
     1201 tatccggaga gctggatcag gacgttttgc taccgctttg ggaaatgcgt gaggaagcgg
     1261 tgaaggggat tacctgcatc gatcttagcc gtgtctcccg cgtggatacg gggggactgg
     1321 cactgctgct ccatcttatc gatctggcga aaaagcaggg caacaatgtg acgcttcagg
     1381 gggtaaacga caaagtgtat accctggcga aattgtataa tttgcctgct gatgttctgc
     1441 ctcgttaaga gagaccggcc aggccggccg atccggctgc taacaaagcc cgaaaggaag
     1501 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     1561 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
     1621 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     1681 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     1741 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     1801 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     1861 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     1921 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     1981 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     2041 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aacgacgacc
     2101 tgcagccaag ctagcttggc tggacgtaaa ctcctcttca gacctaataa cttcgtatag
     2161 catacattat acgaagttat attaagggtt attgaatatg atcggaattg ggctgcagga
     2221 attcgatagc ttggctgcag gtcgacctcg aggatgtgcg cggaacccct atttgtttat
     2281 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     2341 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     2401 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     2461 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     2521 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     2581 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     2641 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     2701 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     2761 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     2821 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     2881 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa
     2941 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     3001 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     3061 ctggagccgg tgagcgtggc tctcgcggta tcattgcagc actggggcca gatggtaagc
     3121 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     3181 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     3241 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     3301 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     3361 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     3421 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     3481 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     3541 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     3601 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     3661 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     3721 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     3781 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     3841 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     3901 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     3961 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4021 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     4081 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     4141 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     4201 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     4261 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     4321 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     4381 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     4441 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     4501 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     4561 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     4621 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     4681 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     4741 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     4801 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     4861 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     4921 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     4981 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     5041 gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc
     5101 tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt
     5161 ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg
     5221 aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc
     5281 ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     5341 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     5401 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     5461 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     5521 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     5581 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     5641 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc
     5701 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     5761 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     5821 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     5881 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     5941 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     6001 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     6061 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     6121 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     6181 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     6241 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     6301 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     6361 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     6421 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     6481 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     6541 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
     6601 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     6661 atggtgcatg caaggagatg gcgcccaaca gtcccccg
//
