LOCUS       sequence-300154-        6636 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses (Strep-MlaF_Delta[248-269])-(6xHis-TEV-MlaB).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6636)
  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 6636)
  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: pBEL2082
FEATURES             Location/Qualifiers
     source          1..6636
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     23..40
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    55..197
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(123..142)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     283..305
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(299..490)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1299..2381)
                     /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     2343..2362
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2382..2459)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2665..2684
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2768..2787
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2768..2786
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2787..2811
                     /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             2868..2891
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3643..3660
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3661..3681
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(4040..4058)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4054..4101
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4138..4593
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4225..4244)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4435..4456
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(4669..4702)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4781..4885
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4886..4954,4955..5746)
                     /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(5104..5123)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5917..6505
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6406..6425
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
       61 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatatg gtgcactctc
      121 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
      181 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
      241 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
      301 agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt
      361 ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct
      421 ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct
      481 gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga
      541 tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg
      601 aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc
      661 agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc
      721 atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac
      781 tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc
      841 agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc
      901 aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg
      961 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     1021 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     1081 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     1141 ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg
     1201 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     1261 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     1321 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     1381 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     1441 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     1501 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     1561 tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc
     1621 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     1681 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     1741 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     1801 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     1861 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     1921 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     1981 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     2041 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     2101 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     2161 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     2221 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     2281 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     2341 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     2401 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     2461 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
     2521 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
     2581 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     2641 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
     2701 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc
     2761 gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa
     2821 ataattttgt ttaactttgg cccaggcggc caggagggtc tcacatgtgg agccacccgc
     2881 agttcgaaaa agagcagtct gtggcgaatt tagtcgatat gcgcgatgtc agttttacgc
     2941 gtggcaatcg ctgcatcttc gataatattt ccctgaccgt gccgcgaggg aagatcacgg
     3001 cgatcatggg gccatcgggc atcggtaaaa cgacgctact ccgtctgatt ggcgggcaaa
     3061 tcgcaccaga tcatggtgag atccttttcg atggtgagaa tattccggcg atgtctcgtt
     3121 cgcgcctgta tacagtgcgc aaacggatga gcatgttatt tcagtccggg gcgttgttca
     3181 ctgatatgaa cgtatttgac aacgtcgcct atccactgcg cgaacatacc caacttcccg
     3241 cgccattgtt gcatagtacg gtgatgatga agctggaggc cgtggggctg cgtggagcgg
     3301 ctaaactaat gccttctgaa ctttccggtg ggatggcgcg gcgtgcagcg ctggcgcgtg
     3361 cgattgcgct ggagccggat ctcatcatgt ttgatgaacc ttttgttggg caagatccca
     3421 tcaccatggg cgtactggtg aagctgattt ctgagctgaa cagcgcgctg ggcgtgactt
     3481 gtgtggtggt ttctcacgat gtgccggaag tgttaagtat tgcggatcac gcctggatcc
     3541 tggcggacaa aaaaattgtc gctcatggca gtgcccaggc gttgcaggcg aatcctgatc
     3601 cgcgcgtacg tcagtttctg gacgggatat aagccactca tgcatcacca ccatcaccat
     3661 gaaaatctgt attttcagag cgagtcactg agctggatgc agacgggtga cacgctggcg
     3721 ttatccggag agctggatca ggacgttttg ctaccgcttt gggaaatgcg tgaggaagcg
     3781 gtgaagggga ttacctgcat cgatcttagc cgtgtctccc gcgtggatac ggggggactg
     3841 gcactgctgc tccatcttat cgatctggcg aaaaagcagg gcaacaatgt gacgcttcag
     3901 ggggtaaacg acaaagtgta taccctggcg aaattgtata atttgcctgc tgatgttctg
     3961 cctcgttaag agagaccggc caggccggcc gatccggctg ctaacaaagc ccgaaaggaa
     4021 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     4081 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg
     4141 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     4201 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     4261 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     4321 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     4381 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     4441 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     4501 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     4561 cgaattttaa caaaatatta acgtttacaa tttcaggtgg cacttttcgg ggaacgacga
     4621 cctgcagcca agctagcttg gctggacgta aactcctctt cagacctaat aacttcgtat
     4681 agcatacatt atacgaagtt atattaaggg ttattgaata tgatcggaat tgggctgcag
     4741 gaattcgata gcttggctgc aggtcgacct cgaggatgtg cgcggaaccc ctatttgttt
     4801 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     4861 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     4921 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     4981 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     5041 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     5101 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     5161 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     5221 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     5281 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     5341 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     5401 aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt
     5461 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     5521 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     5581 atctggagcc ggtgagcgtg gctctcgcgg tatcattgca gcactggggc cagatggtaa
     5641 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     5701 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     5761 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     5821 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     5881 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     5941 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6001 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6061 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6121 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6181 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6241 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6301 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     6361 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     6421 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     6481 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     6541 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     6601 ccgtattacc gcctttgagt gagctgatac cgctcg
//
