LOCUS       sequence-350399-        5794 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Rok with a C-terminal His-tag overexpression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5794)
  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 5794)
  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: pRD461
FEATURES             Location/Qualifiers
     source          1..5794
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      80..535
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(167..186)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     377..398
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(627..1442)
                     /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(627..1442)
                     /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     1350..1369
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1434..1453
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1564..2152
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2053..2072
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2306..2323
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2338..2477
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2403..2422)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2563..2585
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2579..2770)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3579..4661)
                     /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     4623..4642
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4662..4739)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4945..4964
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5052..5071
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5052..5070
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5071..5095
                     /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             5110..5132
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5713..5730
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5776..5794)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      join(5790..5794,1..43)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
       61 atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
      121 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
      181 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
      241 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
      301 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
      361 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
      421 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
      481 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
      541 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
      601 atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc
      661 aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa
      721 ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt
      781 ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca
      841 agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt
      901 tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca
      961 accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta
     1021 aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca
     1081 acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg
     1141 atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga
     1201 agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca
     1261 acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga
     1321 tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca
     1381 gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc
     1441 ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat
     1501 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     1561 ttcttgaaat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     1621 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     1681 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     1741 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     1801 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     1861 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     1921 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     1981 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     2041 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     2101 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     2161 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     2221 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     2281 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     2341 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc
     2401 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
     2461 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     2521 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     2581 agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt
     2641 ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct
     2701 ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct
     2761 gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga
     2821 tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg
     2881 aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc
     2941 agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc
     3001 atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac
     3061 tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc
     3121 agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc
     3181 aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg
     3241 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     3301 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     3361 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     3421 ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg
     3481 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     3541 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     3601 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     3661 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     3721 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     3781 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     3841 tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt aatggcgcgc
     3901 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     3961 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     4021 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     4081 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     4141 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     4201 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     4261 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     4321 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     4381 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     4441 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     4501 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     4561 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     4621 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     4681 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     4741 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
     4801 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
     4861 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     4921 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
     4981 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tcgatctcga
     5041 tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct
     5101 agaaataatt ttgtttaact ttaagaagga gatatacata tgtttaatga aagagaagct
     5161 ttgcgcttgc ggttagaaca attaaatgaa gctgaagtga aagtcattcg tgaatatcaa
     5221 attgaacgtg ataaaatata cgcaaaatta agagagctgg acagaaatgg aagcccttcc
     5281 gaaatcaaaa aggatttccg ttctgaaaaa aaacctgact ctctgccggt tctcgctgag
     5341 cttgcggctc aggaaatcag aagctatcag ccgcaatcac agcagcagtc tgttcagcct
     5401 cagcttcaat ctatttcctc tcttcctgcc ggtataccag acggaacaac acgaagaaga
     5461 agaggaacgg caagaccggg atcaaaagca gctaagctgc gtgaagctgc tattaaaaca
     5521 ttaaaacgcc acaacgcggc gattaagagc tcggagcttc aaaaagagat tgaaaaagaa
     5581 agcggtcttg aaatccctaa tatgacaacg tttatgcaaa gcttaattaa aatgtacccg
     5641 gaagtgaaaa agccatatcg cgggcagtac attttagagg gtgaaatcga atctgcagaa
     5701 tcagcaaacg aacatcatca tcatcatcat taactgctaa caaagcccga aaggaagctg
     5761 agttggctgc tgccaccgct gagcaataac tagc
//
