LOCUS       sequence-318454-        6698 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses (Strep-MlaF_Y261A_L265A_L266A)-(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: pBEL2081
FEATURES             Location/Qualifiers
     source          1..6698
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      81..536
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(168..187)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     378..399
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(611..644)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        723..827
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(828..896,897..1688)
                     /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(1046..1065)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1859..2447
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2348..2367
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2601..2618
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2633..2772
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2698..2717)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2858..2880
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2874..3065)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3874..4956)
                     /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     4918..4937
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4957..5034)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5240..5259
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5343..5362
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5343..5361
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5362..5386
                     /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             5443..5466
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             6284..6301
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6302..6322
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(join(6681..6698,1))
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      join(6695..6698,1..44)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
       61 tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
      121 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
      181 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
      241 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
      301 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
      361 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
      421 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
      481 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt
      541 ggcacttttc ggggaacgac gacctgcagc caagctagct tggctggacg taaactcctc
      601 ttcagaccta ataacttcgt atagcataca ttatacgaag ttatattaag ggttattgaa
      661 tatgatcgga attgggctgc aggaattcga tagcttggct gcaggtcgac ctcgaggatg
      721 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
      781 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
      841 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
      901 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
      961 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     1021 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     1081 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     1141 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     1201 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     1261 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     1321 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg
     1381 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     1441 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     1501 ctggctggtt tattgctgat aaatctggag ccggtgagcg tggctctcgc ggtatcattg
     1561 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     1621 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     1681 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     1741 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     1801 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     1861 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     1921 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     1981 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     2041 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     2101 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     2161 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     2221 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     2281 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     2341 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     2401 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     2461 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     2521 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     2581 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     2641 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac
     2701 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     2761 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     2821 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     2881 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     2941 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     3001 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     3061 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     3121 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     3181 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     3241 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     3301 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     3361 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     3421 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     3481 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc
     3541 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
     3601 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
     3661 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
     3721 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
     3781 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     3841 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3901 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3961 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     4021 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     4081 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     4141 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     4201 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     4261 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     4321 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     4381 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     4441 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     4501 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     4561 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     4621 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     4681 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     4741 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     4801 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     4861 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     4921 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     4981 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     5041 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     5101 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     5161 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     5221 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     5281 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa
     5341 attaatacga ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat
     5401 tttgtttaac tttggcccag gcggccagga gggtctcaca tgtggagcca cccgcagttc
     5461 gaaaaagagc agtctgtggc gaatttagtc gatatgcgcg atgtcagttt tacgcgtggc
     5521 aatcgctgca tcttcgataa tatttccctg accgtgccgc gagggaagat cacggcgatc
     5581 atggggccat cgggcatcgg taaaacgacg ctactccgtc tgattggcgg gcaaatcgca
     5641 ccagatcatg gtgagatcct tttcgatggt gagaatattc cggcgatgtc tcgttcgcgc
     5701 ctgtatacag tgcgcaaacg gatgagcatg ttatttcagt ccggggcgtt gttcactgat
     5761 atgaacgtat ttgacaacgt cgcctatcca ctgcgcgaac atacccaact tcccgcgcca
     5821 ttgttgcata gtacggtgat gatgaagctg gaggccgtgg ggctgcgtgg agcggctaaa
     5881 ctaatgcctt ctgaactttc cggtgggatg gcgcggcgtg cagcgctggc gcgtgcgatt
     5941 gcgctggagc cggatctcat catgtttgat gaaccttttg ttgggcaaga tcccatcacc
     6001 atgggcgtac tggtgaagct gatttctgag ctgaacagcg cgctgggcgt gacttgtgtg
     6061 gtggtttctc acgatgtgcc ggaagtgtta agtattgcgg atcacgcctg gatcctggcg
     6121 gacaaaaaaa ttgtcgctca tggcagtgcc caggcgttgc aggcgaatcc tgatccgcgc
     6181 gtacgtcagt ttctggacgg gatagctgac gggcctgttc cgttccgcta tcctgccggc
     6241 gatgcgcacg ctgatcttgc ggcggggagt taagccactc atgcatcacc accatcacca
     6301 tgaaaatctg tattttcaga gcgagtcact gagctggatg cagacgggtg acacgctggc
     6361 gttatccgga gagctggatc aggacgtttt gctaccgctt tgggaaatgc gtgaggaagc
     6421 ggtgaagggg attacctgca tcgatcttag ccgtgtctcc cgcgtggata cggggggact
     6481 ggcactgctg ctccatctta tcgatctggc gaaaaagcag ggcaacaatg tgacgcttca
     6541 gggggtaaac gacaaagtgt ataccctggc gaaattgtat aatttgcctg ctgatgttct
     6601 gcctcgttaa gagagaccgg ccaggccggc cgatccggct gctaacaaag cccgaaagga
     6661 agctgagttg gctgctgcca ccgctgagca ataactag
//
