LOCUS       sequence-271723-        5400 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Medium 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 5400)
  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 5400)
  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_med
FEATURES             Location/Qualifiers
     source          1..5400
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      107..652
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     553..572
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     769..786
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1435..1451
                     /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(1476..1490,1491..1496,1497..1513,1514..1520)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    1496..1512
                     /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    1526..1545
                     /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(3337..3356)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        3423..3514
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3515..4324
                     /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             join(4407..5400,1..89)
                     /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(4426..4445)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
       61 aggtttcccg actggaaagc gggcagtgat taataagatg atcttcttga gatcgttttg
      121 gtctgcgcgt aatctcttgc tctgaaaacg aaaaaaccgc cttgcagggc ggtttttcga
      181 aggttctctg agctaccaac tctttgaacc gaggtaactg gcttggagga gcgcagtcac
      241 caaaacttgt cctttcagtt tagccttaac cggcgcatga cttcaagact aactcctcta
      301 aatcaattac cagtggctgc tgccagtggt gcttttgcat gtctttccgg gttggactca
      361 agacgatagt taccggataa ggcgcagcgg tcggactgaa cggggggttc gtgcatacag
      421 tccagcttgg agcgaactgc ctacccggaa ctgagtgtca ggcgtggaat gagacaaacg
      481 cggccataac agcggaatga caccggtaaa ccgaaaggca ggaacaggag agcgcacgag
      541 ggagccgcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccaccactg
      601 atttgagcgt cagatttcgt gatgcttgtc aggggggcgg agcctatgga aaaacggctt
      661 tgccgcggcc ctctcacttc cctgttaagt atcttcctgg catcttccag gaaatctccg
      721 ccccgttcgt aagccatttc cgctcgccgc agtcgaacga ccgagcgtag cgagtcagtg
      781 agcgaggaag cggaatatat cctgtatcac atattctgct gacgcaccgg tgcagccttt
      841 tttctcctgc cacatgaagc acttcactga caccctcatc agtgccaaca tagtaagcca
      901 gtatacactc cgctagcgct gaggtcccgc agccgaacga ccgagcgcag cggcgagagt
      961 agggaactgc caggcatcct gggcggttct gataacgagt aatcgttaat ccgcaaataa
     1021 cgtaaaaacc cgcttcggcg ggttttttta tggggggagt ttagggaaag agcatttgtc
     1081 agaatattta agggcgcctg tcactttgct tgatatatga gaattattta accttataaa
     1141 tgagaaaaaa gcaacgcact ttaaataaga tacgttgctt tttcgattga tgaacaccta
     1201 taattaaact attcatctat tatttatgat tttttgtata tacaatattt ctagtttgtt
     1261 aaagagaatt aagaaaataa atctcgaaaa taataaaggg aaaatcagtt tttgatatca
     1321 aaattataca tgtcaacgat aatacaaaat ataatacaaa ctataagatg ttatcagtat
     1381 ttattatgca tttagaatac cttttgtgtc gcccttcgct gaagtcgacc ttaattgtga
     1441 gcggataaca attacgagct tcatgcacag tgaaatcatg aaaaatttat ttgctttgtg
     1501 agcggataac aattataata tgtggaattg tgagcgctca caattccaca acggtttccc
     1561 tctagaaata attttgttta acttttaagg agataaacca tggcacaagt cattaatacc
     1621 aacagcctct cgctgatcac tcaaaataat atcaacaaga accagtctgc gctgtcgagt
     1681 tctatcgagc gtctgtcttc tggcttgcgt attaacagcg cgaaggatga cgcagcgggt
     1741 caggcgattg ctaaccgttt cacctctaac attaaaggcc tgactcaggc ggcccgtaac
     1801 gccaacgacg gtatctccgt tgcgcagacc accgaaggcg cgctgtccga aatcaacaac
     1861 aacttacagc gtgtgcgtga actgacggta caggccacta ccggtactaa ctctgagtct
     1921 gatctgtctt ctatccagga cgaaattaaa tcccgtctgg atgaaattga ccgcgtatct
     1981 ggtcagaccc agttcaacgg cgtgaacgtg ctggcaaaaa atggctccat gaaaatccag
     2041 gttggcgcaa atgataacca gactatcact atcgatctga agcagattga tgctaaaact
     2101 cttggccttg atggttttag cgttaaaaat aacgatacag ttaccactag tgctccagta
     2161 actgcttttg gtgctaccac cacaaacaat attaaactta ctggaattac cctttctacg
     2221 gaagcagcca ctgatactgg cggaactaac ccagcttcaa ttgagggtgt ttatactgat
     2281 aatggtaatg attactatgc gaaaatcacc ggtggtgata acgatgggaa gtattacgca
     2341 gtaacagttg ctaatgatgg tacagtgaca atggcgactg gagcaacggc aaatgcaact
     2401 gtaactgatg caaatactac taaagctaca actatcactt caggcggtac acctgttcag
     2461 attgataata ctgcaggttc cgcaactgcc aaccttggtg ctgttagctt agtaaaactg
     2521 caggattcca agggtaatga taccgataca tatgcgctta aagatacaaa tggcaatctt
     2581 tacgctgcgg atgtgaatga aactactggt gctgtttctg ttaaaactat tacctatact
     2641 gactcttccg gtgccgccag ttctccaacc gcggtcaaac tgggcggaga tgatggcaaa
     2701 acagaagtgg tcgatattga tggtaaaaca tacgattctg ccgatttaaa tggcggtaat
     2761 ctgcaaacag gtttgactgc tggtggtgag gctctgactg ctgttgcaaa tggtaaaacc
     2821 acggatccgc tgaaagcgct ggacgatgct atcgcatctg tagacaaatt ccgttcttcc
     2881 ctcggtgcgg tgcaaaaccg tctggattcc gcggttacca acctgaacaa caccactacc
     2941 aacctgtctg aagcgcagtc ccgtattcag gacgccgact atgcgaccga agtgtccaat
     3001 atgtcgaaag cgcagatcat ccagcaggcc ggtaactccg tgttggcaaa agctaaccag
     3061 gtaccgcagc aggttctgtc tctgctgcag ggttaagaat tctgatcaca ctggctacct
     3121 ttgggtggcc tttttttatc gtcaaaaggg cgacaccccc taattagccc gggcgaaagg
     3181 cccagtcttt cgactgagcc tttcgtttta tttgatgcct ggcagttccc tactctcgca
     3241 tggggagtcc ccacactacc atcggcgcta cggcgtttca cttctgagtt cggcatgggg
     3301 tcaggtggga ccaccgcgct actgccgcca ggcaaacaag gggtgttatg agccatattc
     3361 aggtataaat gggctcgcga taatgttcag aattggttaa ttggttgtaa cactgacccc
     3421 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     3481 ataaatgctt caataatatt gaaaaaggaa gaatatgagc catattcaac gggaaacgtc
     3541 gaggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga
     3601 taatgtcggg caatcaggtg cgacaatcta tcgcttgtat gggaagcccg atgcgccaga
     3661 gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag
     3721 actaaactgg ctgacggaat ttatgccact tccgaccatc aagcatttta tccgtactcc
     3781 tgatgatgca tggttactca ccactgcgat ccccggaaaa acagcgttcc aggtattaga
     3841 agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt
     3901 gcactcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc gcctcgctca
     3961 ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa
     4021 tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga
     4081 ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt
     4141 aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat
     4201 cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata
     4261 tggtattgat aatcctgata tgaataaatt gcaatttcat ttgatgctcg atgagttttt
     4321 ctaagcggcg cgccatcgaa tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg
     4381 aagagagtca attcagggtg gtgaatatga aaccagtaac gttatacgat gtcgcagagt
     4441 atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg
     4501 cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg
     4561 tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg
     4621 ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg
     4681 ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc
     4741 acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg
     4801 atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg
     4861 accagacacc catcaacagt attattttct cccatgagga cggtacgcga ctgggcgtgg
     4921 agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg
     4981 tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc
     5041 cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa
     5101 tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg
     5161 gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg
     5221 gatacgacga taccgaagat agctcatgtt atatcccgcc gttaaccacc atcaaacagg
     5281 attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg
     5341 cggtgaaggg caatcagctg ttgccagtct cactggtgaa aagaaaaacc accctggcgc
//
