LOCUS       sequence-309873-        6698 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Strep-MlaF-(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: pBEL1957
FEATURES             Location/Qualifiers
     source          1..6698
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     522..541
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(561..638)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     844..863
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     947..966
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        947..965
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    966..990
                     /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             1047..1070
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1888..1905
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1906..1926
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(2285..2303)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2299..2346
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2383..2838
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2470..2489)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2680..2701
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(2913..2946)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3025..3129
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3130..3198,3199..3990)
                     /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(3348..3367)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4161..4749
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4650..4669
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4903..4920
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4935..5074
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5000..5019)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5160..5182
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5176..5367)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6176..6698,1..560))
                     /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 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
       61 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
      121 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
      181 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
      241 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
      301 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
      361 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
      421 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
      481 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
      541 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
      601 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
      661 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
      721 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
      781 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
      841 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
      901 cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact
      961 ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttggc
     1021 ccaggcggcc aggagggtct cacatgtgga gccacccgca gttcgaaaaa gagcagtctg
     1081 tggcgaattt agtcgatatg cgcgatgtca gttttacgcg tggcaatcgc tgcatcttcg
     1141 ataatatttc cctgaccgtg ccgcgaggga agatcacggc gatcatgggg ccatcgggca
     1201 tcggtaaaac gacgctactc cgtctgattg gcgggcaaat cgcaccagat catggtgaga
     1261 tccttttcga tggtgagaat attccggcga tgtctcgttc gcgcctgtat acagtgcgca
     1321 aacggatgag catgttattt cagtccgggg cgttgttcac tgatatgaac gtatttgaca
     1381 acgtcgccta tccactgcgc gaacataccc aacttcccgc gccattgttg catagtacgg
     1441 tgatgatgaa gctggaggcc gtggggctgc gtggagcggc taaactaatg ccttctgaac
     1501 tttccggtgg gatggcgcgg cgtgcagcgc tggcgcgtgc gattgcgctg gagccggatc
     1561 tcatcatgtt tgatgaacct tttgttgggc aagatcccat caccatgggc gtactggtga
     1621 agctgatttc tgagctgaac agcgcgctgg gcgtgacttg tgtggtggtt tctcacgatg
     1681 tgccggaagt gttaagtatt gcggatcacg cctggatcct ggcggacaaa aaaattgtcg
     1741 ctcatggcag tgcccaggcg ttgcaggcga atcctgatcc gcgcgtacgt cagtttctgg
     1801 acgggatagc tgacgggcct gttccgttcc gctatcctgc cggcgattat cacgctgatc
     1861 ttttaccagg gagttaagcc actcatgcat caccaccatc accatgaaaa tctgtatttt
     1921 cagagcgagt cactgagctg gatgcagacg ggtgacacgc tggcgttatc cggagagctg
     1981 gatcaggacg ttttgctacc gctttgggaa atgcgtgagg aagcggtgaa ggggattacc
     2041 tgcatcgatc ttagccgtgt ctcccgcgtg gatacggggg gactggcact gctgctccat
     2101 cttatcgatc tggcgaaaaa gcagggcaac aatgtgacgc ttcagggggt aaacgacaaa
     2161 gtgtataccc tggcgaaatt gtataatttg cctgctgatg ttctgcctcg ttaagagaga
     2221 ccggccaggc cggccgatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
     2281 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     2341 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg
     2401 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     2461 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     2521 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     2581 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     2641 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     2701 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     2761 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     2821 tattaacgct tacaatttag gtggcacttt tcggggaacg acgacctgca gccaagctag
     2881 cttggctgga cgtaaactcc tcttcagacc taataacttc gtatagcata cattatacga
     2941 agttatatta agggttattg aatatgatcg gaattgggct gcaggaattc gatagcttgg
     3001 ctgcaggtcg acctcgagga tgtgcgcgga acccctattt gtttattttt ctaaatacat
     3061 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     3121 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     3181 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     3241 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     3301 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     3361 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     3421 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     3481 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     3541 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     3601 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     3661 accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     3721 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     3781 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     3841 cgtggctctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     3901 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     3961 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     4021 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     4081 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     4141 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     4201 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     4261 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     4321 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     4381 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     4441 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     4501 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     4561 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     4621 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     4681 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     4741 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     4801 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     4861 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     4921 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     4981 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     5041 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     5101 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     5161 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     5221 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     5281 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     5341 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     5401 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     5461 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     5521 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     5581 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     5641 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     5701 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     5761 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     5821 catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt
     5881 ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag
     5941 cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag
     6001 cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac
     6061 gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     6121 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     6181 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     6241 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     6301 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     6361 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     6421 agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg
     6481 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     6541 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     6601 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     6661 cagccagacg cagacgcgcc gagacagaac ttaatggg
//
