LOCUS       sequence-421903-        5653 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Xenopus laevis FLAG5-His10-TEV-Cdc45.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5653)
  AUTHORS   Cvetkovic MA, Passaretti P, Butryn A, Reynolds-Winczura A, Kingsley 
            G, Skagia A, Fernandez-Cuesta C, Poovathumkadavil D, George R, 
            Chauhan AS, Jhujh SS, Stewart GS, Gambus A, Costa A
  TITLE     The structural mechanism of dimeric DONSON in replicative helicase 
            activation.
  JOURNAL   Mol Cell. 2023 Nov 16;83(22):4017-4031.e9. doi: 
            10.1016/j.molcel.2023.09.029. Epub 2023 Oct 10.
  PUBMED    37820732
REFERENCE   2  (bases 1 to 5653)
  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: 
            "10.1016/j.molcel.2023.09.029"; journalName: "Mol Cell"; date: 
            "2023-11-16- 16"; volume: "83"; issue: "22"; pages: "4017-4031.e9"
COMMENT     Sequence Label: pRSFDuet-1-Cdc45
FEATURES             Location/Qualifiers
     source          1..5653
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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)."
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        214..232
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    233..257
                     /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)."
     CDS             2004..2024
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2025..2054
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             2055..2126
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     CDS             2079..2150
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     CDS             2103..2174
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     CDS             2190..2234
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2272..2290)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2286..2333
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2563..3378)
                     /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(2563..3378)
                     /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     3286..3305
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3379..3470)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3486..4235)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(3496..3513)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3736..3755)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4431..5513)
                     /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     5475..5494
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5514..5591)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(5637..5653,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgaattcgag
      121 ctcggcgcgc ctgcaggtcg acaagcttgc ggccgcataa tgcttaagtc gaacagaaag
      181 taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt
      241 gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata
      301 tgtttgttag cgatctgcgc aaagaatttt tcgatgttat tgttaccgaa cgtgttctgc
      361 tgctggttgc accggatgtt gatgcactgt gtgcatgtaa aattctgcag gcactgtttc
      421 agtgtgatca tgttcagtat accctggttc cggttagcgg ttggcaagaa ctggaaaccc
      481 tgtttctgga acataaagaa cagtttcgct attttgtgct gattaattgc ggtgccaata
      541 tcgatctgct ggaaacactg cagccgcaag aagaagcaat tttctatatt tgtgataccc
      601 accgtccgat tgatgtggtg aatatctata atgatagcca ggtgaaactg ctgattcgtc
      661 aggatgatga tctggaaatt cctgcctatg atgatatctt caacgatgat gaagaagatg
      721 gcgaagatag cggtaatgaa agtgatggtg cagaaccgag cggtaaacgt cgtcgttttg
      781 atgaagcagc agttgaacgt cgtattgaac gccgtcgtca gcgtcgtgaa tgggaagccc
      841 gtcgtcgtga aattatcttt gattatgagc agtatgagta tcatggcacc agcagcgcaa
      901 tgatgatgtt tgaactggca tggatcatga gcaaagatag caatgatatg ctgtggtggg
      961 caattgttgg tctgaccgat cagtgggttc aggatcgtat tacccagatg aaatatgtta
     1021 ccgatgttgg caccctgcag cgtcatgtta gccgtcataa tcatcgtaat gaagatgaag
     1081 aaaacagcct gagcattgat tgtatgcgta tcgcctttga atatgatctg cgtctgagcc
     1141 tgtatcagca ttggagtctg tatgaaagca tttgcaatag ctgttatacc agcgcaaccc
     1201 tgaaactgtg gtcactgcag ggtcagaaaa aactgcaaga atttctggca gatatgggta
     1261 tgccgctgaa acaggttaaa cagaaattta acagcatgga tatcagcctg aaagaaaatc
     1321 tgcgtgaaat gctggaagaa agcgcaaaca aatttggcat gaaagatgtt cgtgttcaga
     1381 cctttagcgt gcagtttggc tttaaaaaca aattcctggc cagcgatatt gtgtttgcag
     1441 ttctgagcct gctggaaaat accgaacgtg atgaaaaagg caccgacaat tttatcaaag
     1501 cactggatag cctgagccgt agcaatctgg ataaactgca taccggtctg gaaatgggta
     1561 aaaaactgct gtgtgcaatt cagcagaccg ttgcaagctg tatttgtacc aatctgattc
     1621 tgagccaggg tccgtttctg tattgttatc tgatggaagg tacaccggat gtgaaaatgt
     1681 ttagcaatcc gattagcctg tgtctgctgt gtaaatatct gctgaaaagc tttgtttgca
     1741 gcaccaaaaa caaacgttgt aaactgctgc cgctggttct ggcagcaccg ctggatgcag
     1801 aaaaaggtac agttattatg gttggtattc cgcctgaagc agaaagcagc gacaaaaaaa
     1861 acttttttgg tcgtgccttt gaaaaagcag ccgaaagcac cagcagccgt accctgcata
     1921 atcattttga tatgagcatt attgaactgc gcaccgaaga tcgtagcaaa tttctggatg
     1981 cactgattag tctgctgagt gccgaaaatc tgtattttca gagtcatcat caccaccatc
     2041 atcaccatca ccacgattat aaagatgacg acgataagga ttataaagat gatgatgata
     2101 aggattataa agatgacgac gataaggatt ataaagatga tgatgataaa gattataaag
     2161 atgatgatga taaataactc gagtctggta aagaaaccgc tgctgcgaaa tttgaacgcc
     2221 agcacatgga ctcgtctact agcgcagctt aattaaccta ggctgctgcc accgctgagc
     2281 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaac
     2341 ctcaggcatt tgagaagcac acggtcacac tgcttccggt agtcaataaa ccggtaaacc
     2401 agcaatagac ataagcggct atttaacgac cctgccctga accgacgaca agctgacgac
     2461 cgggtctccg caagtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     2521 ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag
     2581 catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag
     2641 ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg
     2701 gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc
     2761 aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg
     2821 caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc
     2881 aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa
     2941 tacgcggtcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa
     3001 cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa
     3061 tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa
     3121 atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc
     3181 tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg
     3241 cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt
     3301 atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc
     3361 ccgttgaata tggctcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     3421 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggcatgca
     3481 gcgctcttcc gcttcctcgc tcactgactc gctacgctcg gtcgttcgac tgcggcgagc
     3541 ggtgtcagct cactcaaaag cggtaatacg gttatccaca gaatcagggg ataaagccgg
     3601 aaagaacatg tgagcaaaaa gcaaagcacc ggaagaagcc aacgccgcag gcgtttttcc
     3661 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagccag aggtggcgaa
     3721 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3781 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3841 cgctttctca tagctcacgc tgttggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3901 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3961 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc attggtaact
     4021 gatttagagg actttgtctt gaagttatgc acctgttaag gctaaactga aagaacagat
     4081 tttggtgagt gcggtcctcc aacccactta ccttggttca aagagttggt agctcagcga
     4141 accttgagaa aaccaccgtt ggtagcggtg gtttttcttt atttatgaga tgatgaatca
     4201 atcggtctat caagtcaacg aacagctatt ccgttactct agatttcagt gcaatttatc
     4261 tcttcaaatg tagcacctga agtcagcccc atacgatata agttgtaatt ctcatgttag
     4321 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     4381 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     4441 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4501 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
     4561 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
     4621 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
     4681 tcttcggtat cgtcgtatcc cactaccgag atgtccgcac caacgcgcag cccggactcg
     4741 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
     4801 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
     4861 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
     4921 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
     4981 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
     5041 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
     5101 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
     5161 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
     5221 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
     5281 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
     5341 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
     5401 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
     5461 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     5521 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     5581 tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaaattaat
     5641 acgactcact ata
//
