LOCUS       sequence-356977-        7311 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 6xHis-tagged RepX in E. coli BL21(DE3), kanamycin 
            resistance, optimal growth at 20 degrees..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7311)
  AUTHORS   Arslan S, Khafizov R, Thomas CD, Chemla YR, Ha T
  TITLE     Protein structure. Engineering of a superhelicase through 
            conformational control.
  JOURNAL   Science. 2015 Apr 17;348(6232):344-7. doi: 10.1126/science.aaa0445.
  PUBMED    25883358
REFERENCE   2  (bases 1 to 7311)
  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"; doi: "10.1126/science"; 
            journalName: "Science"; date: "2015-04-17- 17"; volume: "348"; 
            issue: "6232"; pages: "344-7"
COMMENT     Sequence Label: RepX_pET28a(+)
FEATURES             Location/Qualifiers
     source          1..7311
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2176..2193
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2246..2264)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2260..2307
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2344..2799
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2431..2450)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2641..2662
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2891..3706)
                     /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(2891..3706)
                     /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     3614..3633
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3698..3717
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3828..4416
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4317..4336
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4570..4587
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4602..4741
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4667..4686)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4827..4849
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4843..5034)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5843..6925)
                     /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     6887..6906
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6926..7003)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7209..7228
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagccatatg cgtctaaacc ccggccaaca acaagctgtc
      181 gaattcgtta ccggcccctt gctggtgctg gcgggcgcgg gttccggtaa aactcgtgtt
      241 atcaccaata aaatcgccca tctgatccgc ggtagcgggt accaggcgcg gcacattgcg
      301 gcggtgacct ttactaataa agcagcgcgc gagatgaaag agcgtgtagg gcagacgctg
      361 gggcgcaaag aggcgcgtgg gctgatgatc tccactttcc atacgttggg gctggatatc
      421 atcaaacgcg agtatgcggc gcttgggatg aaagcgaact tctcgttgtt tgacgatacc
      481 gatcagcttg ctttgcttaa agagttgacc gaggggctga ttgaagatga caaagttctc
      541 ctgcaacaac tgatttcgac catctctaac tggaagaatg atctcaaaac accgtcccag
      601 gcggcagcaa gtgcgattgg cgagcgggac cgtatttttg cccatgttta tgggctgtat
      661 gatgcacacc tgaaagcctg taacgttctc gacttcgatg atctgatttt attgccgacg
      721 ttgctgctgc aacgcaatga agaagtccgc aagcgctggc agaacaaaat tcgctatctg
      781 ctggtggatg agtatcagga caccaacacc agccagtatg agctggtgaa actgctggtg
      841 ggcagccgcg cgcgctttac cgtggtgggt gacgatgacc agtcgatcta ctcctggcgc
      901 ggtgcacgtc cgcaaaacct ggtgctgctg agtcaggatt ttccggcgct gaaggtgatt
      961 aagcttgagc agaactatcg ctcttccggg cgtattctga aagcggcgaa catcctgatc
     1021 gccaataacc cgcacgtctt tgaaaagcgt ctgttctccg aactgggtta tggcgcggag
     1081 ctaaaagtat taagcgcgaa taacgaagaa catgaggctg agcgcgttac tggcgagctg
     1141 atcgcccatc acttcgtcaa taaaacgcag tacaaagatt acgccattct ttatcgcggt
     1201 aaccatcagt cgcgggtgtt tgaaaaattc ctgatgcaaa accgcatccc gtacaaaata
     1261 tctggtggta cgtcgttttt ctctcgtcct gaaatcaagg acttgctggc ttatctgcgc
     1321 gtgctgacta acccggacga tgactgcgca tttctgcgta tcgttaacac gccgaagcga
     1381 gagattggcc cggctacgct gaaaaagctg ggtgagtggg cgatgacgcg caataaaagc
     1441 atgtttaccg ccagctttga tatgggcctg agtcagacgc ttagcggacg tggttatgaa
     1501 gcattgaccc gcttcactca ctggttggca gaaatccagc gtctggcgga gcgggagccg
     1561 attgccgcgg tgcgtgatct gatccatggc atggattatg aatcctggct gtacgaaaca
     1621 tcgcccagcc cgaaagccgc cgaaatgcgc atgaagaacg tcaaccaact gtttagctgg
     1681 atgacggaga tgctggaagg cagtgaactg gatgagccga tgacgctcac ccaggtggtg
     1741 acgcgcttta ctttgcgcga catgatggag cgtggtgaga gtgaagaaga gctggatcag
     1801 gtgcaactga tgactctcca cgcgtcgaaa gggctggagt ttccttatgt ctacatggtc
     1861 ggtatggaag aagggttttt gccgcaccag agcagcatcg atgaagataa tatcgatgag
     1921 gagcggcggc tggcctatgt cggcattacc cgcgcccaga aggaattgac ctttacgctg
     1981 gctaaagaac gccgtcagta cggcgaactg gtgcgcccgg agccgagccg ctttttgctg
     2041 gagctgccgc aggatgatct gatttgggaa caggagcgca aagtggtcag cgccgaagaa
     2101 cggatgcaga aagggcaaag ccatctggcg aatctgaaag cgatgatggc ggcaaaacga
     2161 gggaaataac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
     2221 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     2281 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     2341 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     2401 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     2461 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     2521 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     2581 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     2641 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     2701 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     2761 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     2821 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2881 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     2941 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     3001 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     3061 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     3121 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
     3181 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     3241 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa
     3301 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     3361 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     3421 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     3481 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     3541 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     3601 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     3661 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
     3721 tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt
     3781 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     3841 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     3901 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     3961 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4021 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4081 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4141 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4201 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4261 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4321 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4381 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4441 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     4501 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     4561 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     4621 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     4681 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     4741 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     4801 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     4861 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     4921 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     4981 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     5041 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     5101 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     5161 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     5221 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     5281 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     5341 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     5401 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     5461 ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat
     5521 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag
     5581 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa
     5641 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
     5701 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
     5761 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     5821 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     5881 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     5941 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     6001 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     6061 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     6121 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     6181 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     6241 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     6301 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     6361 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     6421 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     6481 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     6541 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     6601 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     6661 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     6721 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     6781 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     6841 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     6901 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     6961 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     7021 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     7081 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     7141 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     7201 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
     7261 ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa t
//
