LOCUS       sequence-271727-        6855 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Low copy number plasmid for expression of E. coli flagellin from an 
            IPTG-inducible T5 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6855)
  AUTHORS   Thomson NM, Pallen MJ
  TITLE     Restoration of wild-type motility to flagellin-knockout Escherichia 
            coli by varying promoter, copy number and induction strength in 
            plasmid-based expression of flagellin.
  JOURNAL   Curr Res Biotechnol. 2020 Nov;2:45-52. doi: 
            10.1016/j.crbiot.2020.03.001.
  PUBMED    33381753
REFERENCE   2  (bases 1 to 6855)
  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: "Curr Res 
            Biotechnol."; date: "2020-11"; volume: "2:45-52. doi"; pages: " 
            10.1016/j.crbiot.2020.03.001"
COMMENT     Sequence Label: pJ291_WT_low
FEATURES             Location/Qualifiers
     source          1..6855
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    193..209
                     /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)."
     promoter        join(234..248,249..254,255..271,272..278)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    254..270
                     /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)."
     protein_bind    284..303
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     primer_bind     complement(2095..2114)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        2181..2272
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2273..3082
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             3165..4247
                     /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     complement(3184..3203)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     terminator      4261..4332
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4348..4375
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(4438..5586)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     IVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     rep_origin      5979..6510
                     /label=oriV
                     /note="incP origin of replication"
ORIGIN
        1 caatatttct agtttgttaa agagaattaa gaaaataaat ctcgaaaata ataaagggaa
       61 aatcagtttt tgatatcaaa attatacatg tcaacgataa tacaaaatat aatacaaact
      121 ataagatgtt atcagtattt attatgcatt tagaatacct tttgtgtcgc ccttcgctga
      181 agtcgacctt aattgtgagc ggataacaat tacgagcttc atgcacagtg aaatcatgaa
      241 aaatttattt gctttgtgag cggataacaa ttataatatg tggaattgtg agcgctcaca
      301 attccacaac ggtttccctc tagaaataat tttgtttaac ttttaaggaa gtaaaccatg
      361 gcacaagtca ttaataccaa cagcctctcg ctgatcactc aaaataatat caacaagaac
      421 cagtctgcgc tgtcgagttc tatcgagcgt ctgtcttctg gcttgcgtat taacagcgcg
      481 aaggatgacg cagcgggtca ggcgattgct aaccgtttca cctctaacat taaaggcctg
      541 actcaggcgg cccgtaacgc caacgacggt atctccgttg cgcagaccac cgaaggcgcg
      601 ctgtccgaaa tcaacaacaa cttacagcgt gtgcgtgaac tgacggtaca ggccactacc
      661 ggtactaact ctgagtctga tctgtcttct atccaggacg aaattaaatc ccgtctggat
      721 gaaattgacc gcgtatctgg tcagacccag ttcaacggcg tgaacgtgct ggcaaaaaat
      781 ggctccatga aaatccaggt tggcgcaaat gataaccaga ctatcactat cgatctgaag
      841 cagattgatg ctaaaactct tggccttgat ggttttagcg ttaaaaataa cgatacagtt
      901 accactagtg ctccagtaac tgcttttggt gctaccacca caaacaatat taaacttact
      961 ggaattaccc tttctacgga agcagccact gatactggcg gaactaaccc agcttcaatt
     1021 gagggtgttt atactgataa tggtaatgat tactatgcga aaatcaccgg tggtgataac
     1081 gatgggaagt attacgcagt aacagttgct aatgatggta cagtgacaat ggcgactgga
     1141 gcaacggcaa atgcaactgt aactgatgca aatactacta aagctacaac tatcacttca
     1201 ggcggtacac ctgttcagat tgataatact gcaggttccg caactgccaa ccttggtgct
     1261 gttagcttag taaaactgca ggattccaag ggtaatgata ccgatacata tgcgcttaaa
     1321 gatacaaatg gcaatcttta cgctgcggat gtgaatgaaa ctactggtgc tgtttctgtt
     1381 aaaactatta cctatactga ctcttccggt gccgccagtt ctccaaccgc ggtcaaactg
     1441 ggcggagatg atggcaaaac agaagtggtc gatattgatg gtaaaacata cgattctgcc
     1501 gatttaaatg gcggtaatct gcaaacaggt ttgactgctg gtggtgaggc tctgactgct
     1561 gttgcaaatg gtaaaaccac ggatccgctg aaagcgctgg acgatgctat cgcatctgta
     1621 gacaaattcc gttcttccct cggtgcggtg caaaaccgtc tggattccgc ggttaccaac
     1681 ctgaacaaca ccactaccaa cctgtctgaa gcgcagtccc gtattcagga cgccgactat
     1741 gcgaccgaag tgtccaatat gtcgaaagcg cagatcatcc agcaggccgg taactccgtg
     1801 ttggcaaaag ctaaccaggt accgcagcag gttctgtctc tgctgcaggg ttaagaattc
     1861 tgatcacact ggctaccttt gggtggcctt tttttatcgt caaaagggcg acacccccta
     1921 attagcccgg gcgaaaggcc cagtctttcg actgagcctt tcgttttatt tgatgcctgg
     1981 cagttcccta ctctcgcatg gggagtcccc acactaccat cggcgctacg gcgtttcact
     2041 tctgagttcg gcatggggtc aggtgggacc accgcgctac tgccgccagg caaacaaggg
     2101 gtgttatgag ccatattcag gtataaatgg gctcgcgata atgttcagaa ttggttaatt
     2161 ggttgtaaca ctgaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     2221 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga atatgagcca
     2281 tattcaacgg gaaacgtcga ggccgcgatt aaattccaac atggatgctg atttatatgg
     2341 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gcttgtatgg
     2401 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     2461 tacagatgag atggtcagac taaactggct gacggaattt atgccacttc cgaccatcaa
     2521 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccggaaaaac
     2581 agcgttccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     2641 agtgttcctg cgccggttgc actcgattcc tgtttgtaat tgtcctttta acagcgatcg
     2701 cgtatttcgc ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     2761 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact
     2821 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     2881 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     2941 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     3001 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc aatttcattt
     3061 gatgctcgat gagtttttct aagcggcgcg ccatcgaatg gcgcaaaacc tttcgcggta
     3121 tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatatgaaa ccagtaacgt
     3181 tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc
     3241 aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga
     3301 attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg
     3361 ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc
     3421 gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag
     3481 cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact
     3541 atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt
     3601 tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc catgaggacg
     3661 gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag
     3721 cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca
     3781 ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt
     3841 ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca
     3901 acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg
     3961 cggatatctc ggtagtggga tacgacgata ccgaagatag ctcatgttat atcccgccgt
     4021 taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc
     4081 aactctctca gggccaggcg gtgaagggca atcagctgtt gccagtctca ctggtgaaaa
     4141 gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     4201 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgaagc ttccaggcat
     4261 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg
     4321 gtgaacgctc tctactagag tcacactggc tcaccttcgg gtgggccttt ctgcgtttat
     4381 acctagggcg atgcaggtgg ctgctgaacc cccagccgga actgacccca caaggcccta
     4441 gcgtttgcaa tgcaccaggt catcattgac ccaggcgtgt tccaccaggc cgctgcctcg
     4501 caactcttcg caggcttcgc cgacctgctc gcgccacttc ttcacgcggg tggaatccga
     4561 tccgcacatg aggcggaagg tttccagctt gagcgggtac ggctcccggt gcgagctgaa
     4621 atagtcgaac atccgtcggg ccgtcggcga cagcttgcgg tacttctccc atatgaattt
     4681 cgtgtagtgg tcgccagcaa acagcacgac gatttcctcg tcgatcagga cctggcaacg
     4741 ggacgttttc ttgccacggt ccaggacgcg gaagcggtgc agcagcgaca ccgattccag
     4801 gtgcccaacg cggtcggacg tgaagcccat cgccgtcgcc tgtaggcgcg acaggcattc
     4861 ctcggccttc gtgtaatacc ggccattgat cgaccagccc aggtcctggc aaagctcgta
     4921 gaacgtgaag gtgatcggct cgccgatagg ggtgcgcttc gcgtactcca acacctgctg
     4981 ccacaccagt tcgtcatcgt cggcccgcag ctcgacgccg gtgtaggtga tcttcacgtc
     5041 cttgttgacg tggaaaatga ccttgttttg cagcgcctcg cgcgggattt tcttgttgcg
     5101 cgtggtgaac agggcagagc gggccgtgtc gtttggcatc gctcgcatcg tgtccggcca
     5161 cggcgcaata tcgaacaagg aaagctgcat ttccttgatc tgctgcttcg tgtgtttcag
     5221 caacgcggcc tgcttggctt cgctgacctg ttttgccagg tcctcgccgg cggtttttcg
     5281 cttcttggtc gtcatagttc ctcgcgtgtc gatggtcatc gacttcgcca aacctgccgc
     5341 ctcctgttcg agacgacgcg aacgctccac ggcggccgat ggcgcgggca gggcaggggg
     5401 agccagttgc acgctgtcgc gctcgatctt ggccgtagct tgctggacta tcgagccgac
     5461 ggactggaag gtttcgcggg gcgcacgcat gacggtgcgg cttgcgatgg tttcggcatc
     5521 ctcggcggaa aaccccgcgt cgatcagttc ttgcctgtat gccttccggt caaacgtccg
     5581 attcattcac cctccttgcg ggattgcccc ggaattaatt ccccggatcg atccgtcgat
     5641 cttgatcccc tgcgccatca gatccttggc ggcaagaaag ccatccagtt tactttgcag
     5701 ggcttcccaa ccttaccaga gggcgcccca gctggcaatt ccggttcgct tgctgtccat
     5761 aaaaccgccc agtctagcta tcgccatgta agcccactgc aagctacctg ctttctcttt
     5821 gcgcttgcgt tttcccttgt ccagatagcc cagtagctga cattcatccg gggtcagcac
     5881 cgtttctgcg gactggcttt ctacgtggct gccatttttg gggtgaggcc gttcgcggcc
     5941 gaggggcgca gcccctgggg ggatgggagg cccgcgttag cgggccggga gggttcgaga
     6001 agggggggca ccccccttcg gcgtgcgcgg tcacgcgcac agggcgcagc cctggttaaa
     6061 aacaaggttt ataaatattg gtttaaaagc aggttaaaag acaggttagc ggtggccgaa
     6121 aaacgggcgg aaacccttgc aaatgctgga ttttctgcct gtggacagcc cctcaaatgt
     6181 caataggtgc gcccctcatc tgtcagcact ctgcccctca agtgtcaagg atcgcgcccc
     6241 tcatctgtca gtagtcgcgc ccctcaagtg tcaataccgc agggcactta tccccaggct
     6301 tgtccacatc atctgtggga aactcgcgta aaatcaggcg ttttcgccga tttgcgaggc
     6361 tggccagctc cacgtcgccg gccgaaatcg agcctgcccc tcatctgtca acgccgcgcc
     6421 gggtgagtcg gcccctcaag tgtcaacgtc cgcccctcat ctgtcagtga gggccaagtt
     6481 ttccgcgagg tatccacaac gccggcggcc ctacatggct ctgcgctagc gctgaggtcc
     6541 cgcagccgaa cgaccgagcg cagcggcgag agtagggaac tgccaggcat cctgggcggt
     6601 tctgataacg agtaatcgtt aatccgcaaa taacgtaaaa acccgcttcg gcgggttttt
     6661 ttatgggggg agtttaggga aagagcattt gtcagaatat ttaagggcgc ctgtcacttt
     6721 gcttgatata tgagaattat ttaaccttat aaatgagaaa aaagcaacgc actttaaata
     6781 agatacgttg ctttttcgat tgatgaacac ctataattaa actattcatc tattatttat
     6841 gattttttgt atata
//
