LOCUS       sequence-309895-        6698 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses Strep-MlaF-(6xHis-TEV-MlaB_T52E).
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 Apr 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: pBEL2074
FEATURES             Location/Qualifiers
     source          1..6698
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     28..47
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     131..150
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        131..149
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    150..174
                     /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             231..254
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1072..1089
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1090..1110
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(1469..1487)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1483..1530
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1567..2022
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1654..1673)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1864..1885
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(2097..2130)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2209..2313
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2314..2382,2383..3174)
                     /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(2532..2551)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3345..3933
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3834..3853
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4087..4104
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4119..4258
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4184..4203)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4344..4366
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4360..4551)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5360..6442)
                     /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     6404..6423
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6443..6520)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
       61 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat
      121 cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta
      181 gaaataattt tgtttaactt tggcccaggc ggccaggagg gtctcacatg tggagccacc
      241 cgcagttcga aaaagagcag tctgtggcga atttagtcga tatgcgcgat gtcagtttta
      301 cgcgtggcaa tcgctgcatc ttcgataata tttccctgac cgtgccgcga gggaagatca
      361 cggcgatcat ggggccatcg ggcatcggta aaacgacgct actccgtctg attggcgggc
      421 aaatcgcacc agatcatggt gagatccttt tcgatggtga gaatattccg gcgatgtctc
      481 gttcgcgcct gtatacagtg cgcaaacgga tgagcatgtt atttcagtcc ggggcgttgt
      541 tcactgatat gaacgtattt gacaacgtcg cctatccact gcgcgaacat acccaacttc
      601 ccgcgccatt gttgcatagt acggtgatga tgaagctgga ggccgtgggg ctgcgtggag
      661 cggctaaact aatgccttct gaactttccg gtgggatggc gcggcgtgca gcgctggcgc
      721 gtgcgattgc gctggagccg gatctcatca tgtttgatga accttttgtt gggcaagatc
      781 ccatcaccat gggcgtactg gtgaagctga tttctgagct gaacagcgcg ctgggcgtga
      841 cttgtgtggt ggtttctcac gatgtgccgg aagtgttaag tattgcggat cacgcctgga
      901 tcctggcgga caaaaaaatt gtcgctcatg gcagtgccca ggcgttgcag gcgaatcctg
      961 atccgcgcgt acgtcagttt ctggacggga tagctgacgg gcctgttccg ttccgctatc
     1021 ctgccggcga ttatcacgct gatcttttac cagggagtta agccactcat gcatcaccac
     1081 catcaccatg aaaatctgta ttttcagagc gagtcactga gctggatgca gacgggtgac
     1141 acgctggcgt tatccggaga gctggatcag gacgttttgc taccgctttg ggaaatgcgt
     1201 gaggaagcgg tgaaggggat tacctgcatc gatcttagcc gtgtctcccg cgtggatgaa
     1261 gggggactgg cactgctgct ccatcttatc gatctggcga aaaagcaggg caacaatgtg
     1321 acgcttcagg gggtaaacga caaagtgtat accctggcga aattgtataa tttgcctgct
     1381 gatgttctgc ctcgttaaga gagaccggcc aggccggccg atccggctgc taacaaagcc
     1441 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     1501 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
     1561 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     1621 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     1681 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     1741 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     1801 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     1861 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     1921 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     1981 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     2041 aacgacgacc tgcagccaag ctagcttggc tggacgtaaa ctcctcttca gacctaataa
     2101 cttcgtatag catacattat acgaagttat attaagggtt attgaatatg atcggaattg
     2161 ggctgcagga attcgatagc ttggctgcag gtcgacctcg aggatgtgcg cggaacccct
     2221 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     2281 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     2341 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     2401 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     2461 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     2521 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     2581 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     2641 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     2701 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     2761 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     2821 gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc
     2881 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     2941 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     3001 gctgataaat ctggagccgg tgagcgtggc tctcgcggta tcattgcagc actggggcca
     3061 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     3121 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     3181 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     3241 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     3301 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3361 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     3421 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     3481 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     3541 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     3601 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3661 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3721 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     3781 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     3841 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     3901 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     3961 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     4021 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     4081 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     4141 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg
     4201 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     4261 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     4321 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     4381 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca
     4441 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     4501 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     4561 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     4621 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     4681 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     4741 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     4801 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
     4861 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
     4921 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
     4981 ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat
     5041 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc
     5101 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg
     5161 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa
     5221 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac
     5281 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     5341 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     5401 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     5461 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     5521 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     5581 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     5641 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     5701 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     5761 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     5821 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     5881 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     5941 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     6001 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     6061 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     6121 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     6181 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     6241 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     6301 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     6361 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     6421 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     6481 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     6541 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     6601 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     6661 gccaccatac ccacgccgaa acaagcgctc atgagccc
//
