LOCUS       sequence-350128-        5776 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Rok overexpression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5776)
  AUTHORS   Erkelens AM, Qin L, van Erp B, Miguel-Arribas A, Abia D, Keek HGJ, 
            Markus D, Cajili MKM, Schwab S, Meijer WJJ, Dame RT
  TITLE     The B. subtilis Rok protein is an atypical H-NS-like protein 
            irresponsive to physico-chemical cues.
  JOURNAL   Nucleic Acids Res. 2022 Nov 21. pii: 6835367. doi: 
            10.1093/nar/gkac1064.
  PUBMED    36408910
REFERENCE   2  (bases 1 to 5776)
  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: "Nucleic 
            Acids Res. 2022 Nov 21. pii: 6835367. doi: 10.1093/nar/gkac1064."
COMMENT     Sequence Label: pRD231
FEATURES             Location/Qualifiers
     source          1..5776
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(145..1227)
                     /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
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTAPPR
                     ALADSLMQLVRQVSRLESGQ"
     primer_bind     1189..1208
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1228..1305)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1511..1530
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1618..1637
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1618..1636
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1637..1661
                     /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)."
     RBS             1676..1698
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(2324..2342)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2338..2385
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2422..2877
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2509..2528)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2719..2740
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2969..3784)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2969..3784)
                     /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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3692..3711
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3776..3795
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3906..4494
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4395..4414
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4648..4665
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4680..4819
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4745..4764)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4905..4927
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4921..5112)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
       61 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
      121 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtac
      181 cagctgcatt aatgaatcgg ccaacgcgcg gggaggggcg gtttgcgtat tgggcgccag
      241 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
      301 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
      361 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
      421 cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
      481 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
      541 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
      601 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
      661 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
      721 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
      781 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
      841 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
      901 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
      961 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgta gggccagact
     1021 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     1081 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     1141 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     1201 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     1261 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     1321 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     1381 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     1441 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     1501 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     1561 tgccggccac gatgcgtccg gcgtagagga tcgagatcga tctcgatccc gcgaaattaa
     1621 tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt
     1681 ttaactttaa gaaggagata tacatatgtt taatgaaaga gaagctttgc gcttgcggtt
     1741 agaacaatta aatgaagctg aagtgaaagt cattcgtgaa tatcaaattg aacgtgataa
     1801 aatatacgca aaattaagag agctggacag aaatggaagc ccttccgaaa tcaaaaagga
     1861 tttccgttct gaaaaaaaac ctgactctct gccggttctc gctgagcttg cggctcagga
     1921 aatcagaagc tatcagccgc aatcacagca gcagtctgtt cagcctcagc ttcaatctat
     1981 ttcctctctt cctgccggta taccagacgg aacaacacga agaagaagag gaacggcaag
     2041 accgggatca aaagcagcta agctgcgtga agctgctatt aaaacattaa aacgccacaa
     2101 cgcggcgatt aagagctcgg agcttcaaaa agagattgaa aaagaaagcg gtcttgaaat
     2161 ccctaatatg acaacgttta tgcaaagctt aattaaaatg tacccggaag tgaaaaagcc
     2221 atatcgcggg cagtacattt tagagggtga aatcgaatct gcggaatcag caaacgaata
     2281 actgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     2341 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     2401 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     2461 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     2521 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     2581 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     2641 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     2701 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     2761 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     2821 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     2881 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     2941 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact
     3001 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     3061 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     3121 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     3181 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
     3241 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     3301 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
     3361 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     3421 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     3481 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     3541 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     3601 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     3661 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     3721 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc
     3781 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa
     3841 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     3901 tcttcttgaa atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     3961 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     4021 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     4081 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     4141 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     4201 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     4261 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     4321 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     4381 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     4441 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     4501 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     4561 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     4621 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     4681 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     4741 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     4801 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     4861 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     4921 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     4981 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     5041 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     5101 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     5161 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     5221 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     5281 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     5341 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     5401 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     5461 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     5521 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
     5581 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     5641 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     5701 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     5761 tcctacgagt tgcatg
//
