LOCUS       sequence-271722-        5471 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  High 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 5471)
  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 5471)
  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: pD441_WT_hi
FEATURES             Location/Qualifiers
     source          1..5471
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      166..754
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     655..674
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     908..925
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1023..1040
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1024..1040
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1497..1513
                     /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(1538..1552,1553..1558,1559..1575,1576..1582)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    1558..1574
                     /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    1588..1607
                     /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."
     RBS             1648..1656
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     primer_bind     complement(3399..3418)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        3485..3576
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3577..4386
                     /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(4469..5471,1..80)
                     /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(4488..4507)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
       61 gactggaaag cgggcagtga ctcatgacca aaatccctta acgtgagtta cgcgcgcgtc
      121 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
      181 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
      241 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
      301 accaaatact gttcttctag tgtagccgta gttagcccac cacttcaaga actctgtagc
      361 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
      421 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
      481 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
      541 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
      601 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
      661 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
      721 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
      781 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
      841 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
      901 cgagcgcagc gagtcagtga gcgaggaagc ggaaggcgag agtagggaac tgccaggcat
      961 caaactaagc agaaggcccc tgacggatgg cctttttgcg tttctacaaa ctctttctgt
     1021 gttgtaaaac gacggccagt cttaagctcg ggccccctgg gcggttctga taacgagtaa
     1081 tcgttaatcc gcaaataacg taaaaacccg cttcggcggg tttttttatg gggggagttt
     1141 agggaaagag catttgtcag aatatttaag ggcgcctgtc actttgcttg atatatgaga
     1201 attatttaac cttataaatg agaaaaaagc aacgcacttt aaataagata cgttgctttt
     1261 tcgattgatg aacacctata attaaactat tcatctatta tttatgattt tttgtatata
     1321 caatatttct agtttgttaa agagaattaa gaaaataaat ctcgaaaata ataaagggaa
     1381 aatcagtttt tgatatcaaa attatacatg tcaacgataa tacaaaatat aatacaaact
     1441 ataagatgtt atcagtattt attatgcatt tagaataaat tttgtgtcgc ccttaattgt
     1501 gagcggataa caattacgag cttcatgcac agtgaaatca tgaaaaattt atttgctttg
     1561 tgagcggata acaattataa tatgtggaat tgtgagcgct cacaattcca caacggtttc
     1621 cctatagaaa taattttgtt taacttttaa ggaggtaaac catggcacaa gtcattaata
     1681 ccaacagcct ctcgctgatc actcaaaata atatcaacaa gaaccagtct gcgctgtcga
     1741 gttctatcga gcgtctgtct tctggcttgc gtattaacag cgcgaaggat gacgcagcgg
     1801 gtcaggcgat tgctaaccgt ttcacctcta acattaaagg cctgactcag gcggcccgta
     1861 acgccaacga cggtatctcc gttgcgcaga ccaccgaagg cgcgctgtcc gaaatcaaca
     1921 acaacttaca gcgtgtgcgt gaactgacgg tacaggccac taccggtact aactctgagt
     1981 ctgatctgtc ttctatccag gacgaaatta aatcccgtct ggatgaaatt gaccgcgtat
     2041 ctggtcagac ccagttcaac ggcgtgaacg tgctggcaaa aaatggctcc atgaaaatcc
     2101 aggttggcgc aaatgataac cagactatca ctatcgatct gaagcagatt gatgctaaaa
     2161 ctcttggcct tgatggtttt agcgttaaaa ataacgatac agttaccact agtgctccag
     2221 taactgcttt tggtgctacc accacaaaca atattaaact tactggaatt accctttcta
     2281 cggaagcagc cactgatact ggcggaacta acccagcttc aattgagggt gtttatactg
     2341 ataatggtaa tgattactat gcgaaaatca ccggtggtga taacgatggg aagtattacg
     2401 cagtaacagt tgctaatgat ggtacagtga caatggcgac tggagcaacg gcaaatgcaa
     2461 ctgtaactga tgcaaatact actaaagcta caactatcac ttcaggcggt acacctgttc
     2521 agattgataa tactgcaggt tccgcaactg ccaaccttgg tgctgttagc ttagtaaaac
     2581 tgcaggattc caagggtaat gataccgata catatgcgct taaagataca aatggcaatc
     2641 tttacgctgc ggatgtgaat gaaactactg gtgctgtttc tgttaaaact attacctata
     2701 ctgactcttc cggtgccgcc agttctccaa ccgcggtcaa actgggcgga gatgatggca
     2761 aaacagaagt ggtcgatatt gatggtaaaa catacgattc tgccgattta aatggcggta
     2821 atctgcaaac aggtttgact gctggtggtg aggctctgac tgctgttgca aatggtaaaa
     2881 ccacggatcc gctgaaagcg ctggacgatg ctatcgcatc tgtagacaaa ttccgttctt
     2941 ccctcggtgc ggtgcaaaac cgtctggatt ccgcggttac caacctgaac aacaccacta
     3001 ccaacctgtc tgaagcgcag tcccgtattc aggacgccga ctatgcgacc gaagtgtcca
     3061 atatgtcgaa agcgcagatc atccagcagg ccggtaactc cgtgttggca aaagctaacc
     3121 aggtaccgca gcaggttctg tctctgctgc agggttaaga attctgatca cactggctac
     3181 ctttgggtgg ccttttttta tcgtcaaaag ggcgacaccc cctaattagc ccgggcgaaa
     3241 ggcccagtct ttcgactgag cctttcgttt tatttgatgc ctggcagttc cctactctcg
     3301 catggggagt ccccacacta ccatcggcgc tacggcgttt cacttctgag ttcggcatgg
     3361 ggtcaggtgg gaccaccgcg ctactgccgc caggcaaaca aggggtgtta tgagccatat
     3421 tcaggtataa atgggctcgc gataatgttc agaattggtt aattggttgt aacactgacc
     3481 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     3541 tgataaatgc ttcaataata ttgaaaaagg aagaatatga gccatattca acgggaaacg
     3601 tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc
     3661 gataatgtcg ggcaatcagg tgcgacaatc tatcgcttgt atgggaagcc cgatgcgcca
     3721 gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc
     3781 agactaaact ggctgacgga atttatgcca cttccgacca tcaagcattt tatccgtact
     3841 cctgatgatg catggttact caccactgcg atccccggaa aaacagcgtt ccaggtatta
     3901 gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg
     3961 ttgcactcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgcctcgct
     4021 caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt
     4081 aatggctggc ctgttgaaca agtctggaaa gaaatgcata aacttttgcc attctcaccg
     4141 gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa
     4201 ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc
     4261 atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa
     4321 tatggtattg ataatcctga tatgaataaa ttgcaatttc atttgatgct cgatgagttt
     4381 ttctaagcgg cgcgccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc
     4441 ggaagagagt caattcaggg tggtgaatat gaaaccagta acgttatacg atgtcgcaga
     4501 gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc
     4561 tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg
     4621 cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct
     4681 ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg
     4741 tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt
     4801 gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca
     4861 ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc
     4921 tgaccagaca cccatcaaca gtattatttt ctcccatgag gacggtacgc gactgggcgt
     4981 ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc
     5041 tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca
     5101 gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca
     5161 aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct
     5221 gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt
     5281 gggatacgac gataccgaag atagctcatg ttatatcccg ccgttaacca ccatcaaaca
     5341 ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca
     5401 ggcggtgaag ggcaatcagc tgttgccagt ctcactggtg aaaagaaaaa ccaccctggc
     5461 gcccaatacg c
//
