LOCUS       sequence-309898-        6698 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses (Strep-MlaF_Y261A)-(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 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: pBEL2078
FEATURES             Location/Qualifiers
     source          1..6698
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     26..45
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     129..148
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        129..147
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    148..172
                     /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             229..252
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1070..1087
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1088..1108
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(1467..1485)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1481..1528
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1565..2020
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1652..1671)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1862..1883
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(2095..2128)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2207..2311
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2312..2380,2381..3172)
                     /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(2530..2549)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3343..3931
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3832..3851
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4085..4102
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4117..4256
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4182..4201)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4342..4364
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4358..4549)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5358..6440)
                     /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     6402..6421
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6441..6518)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
       61 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc
      121 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
      181 aataattttg tttaactttg gcccaggcgg ccaggagggt ctcacatgtg gagccacccg
      241 cagttcgaaa aagagcagtc tgtggcgaat ttagtcgata tgcgcgatgt cagttttacg
      301 cgtggcaatc gctgcatctt cgataatatt tccctgaccg tgccgcgagg gaagatcacg
      361 gcgatcatgg ggccatcggg catcggtaaa acgacgctac tccgtctgat tggcgggcaa
      421 atcgcaccag atcatggtga gatccttttc gatggtgaga atattccggc gatgtctcgt
      481 tcgcgcctgt atacagtgcg caaacggatg agcatgttat ttcagtccgg ggcgttgttc
      541 actgatatga acgtatttga caacgtcgcc tatccactgc gcgaacatac ccaacttccc
      601 gcgccattgt tgcatagtac ggtgatgatg aagctggagg ccgtggggct gcgtggagcg
      661 gctaaactaa tgccttctga actttccggt gggatggcgc ggcgtgcagc gctggcgcgt
      721 gcgattgcgc tggagccgga tctcatcatg tttgatgaac cttttgttgg gcaagatccc
      781 atcaccatgg gcgtactggt gaagctgatt tctgagctga acagcgcgct gggcgtgact
      841 tgtgtggtgg tttctcacga tgtgccggaa gtgttaagta ttgcggatca cgcctggatc
      901 ctggcggaca aaaaaattgt cgctcatggc agtgcccagg cgttgcaggc gaatcctgat
      961 ccgcgcgtac gtcagtttct ggacgggata gctgacgggc ctgttccgtt ccgctatcct
     1021 gccggcgatg cgcacgctga tcttttacca gggagttaag ccactcatgc atcaccacca
     1081 tcaccatgaa aatctgtatt ttcagagcga gtcactgagc tggatgcaga cgggtgacac
     1141 gctggcgtta tccggagagc tggatcagga cgttttgcta ccgctttggg aaatgcgtga
     1201 ggaagcggtg aaggggatta cctgcatcga tcttagccgt gtctcccgcg tggatacggg
     1261 gggactggca ctgctgctcc atcttatcga tctggcgaaa aagcagggca acaatgtgac
     1321 gcttcagggg gtaaacgaca aagtgtatac cctggcgaaa ttgtataatt tgcctgctga
     1381 tgttctgcct cgttaagaga gaccggccag gccggccgat ccggctgcta acaaagcccg
     1441 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     1501 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa
     1561 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     1621 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     1681 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     1741 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     1801 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     1861 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     1921 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     1981 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     2041 cgacgacctg cagccaagct agcttggctg gacgtaaact cctcttcaga cctaataact
     2101 tcgtatagca tacattatac gaagttatat taagggttat tgaatatgat cggaattggg
     2161 ctgcaggaat tcgatagctt ggctgcaggt cgacctcgag gatgtgcgcg gaacccctat
     2221 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     2281 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     2341 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     2401 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     2461 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     2521 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     2581 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     2641 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     2701 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     2761 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     2821 cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa
     2881 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     2941 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     3001 tgataaatct ggagccggtg agcgtggctc tcgcggtatc attgcagcac tggggccaga
     3061 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     3121 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     3181 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     3241 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     3301 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     3361 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     3421 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     3481 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     3541 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     3601 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     3661 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     3721 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     3781 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     3841 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     3901 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     3961 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     4021 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     4081 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     4141 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc
     4201 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     4261 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     4321 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     4381 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     4441 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     4501 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     4561 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     4621 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     4681 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     4741 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     4801 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     4861 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     4921 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     4981 tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg
     5041 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
     5101 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt
     5161 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
     5221 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
     5281 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     5341 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5401 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     5461 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     5521 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     5581 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
     5641 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     5701 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     5761 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     5821 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     5881 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     5941 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     6001 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     6061 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     6121 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     6181 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     6241 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     6301 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     6361 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     6421 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     6481 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     6541 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     6601 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
     6661 caccataccc acgccgaaac aagcgctcat gagcccga
//
