LOCUS       sequence-413082-        4568 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of human Replication Protein A 4 in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4568)
  AUTHORS   Mason AC, Haring SJ, Pryor JM, Staloch CA, Gan TF, Wold MS
  TITLE     An alternative form of replication protein a prevents viral 
            replication in vitro.
  JOURNAL   J Biol Chem. 2009 Feb 20;284(8):5324-31. doi: 
            10.1074/jbc.M808963200. Epub 2008 Dec 29.
  PUBMED    19116208
REFERENCE   2  (bases 1 to 4568)
  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: 
            "10.1074/jbc.M808963200"; journalName: "J Biol Chem"; date: 
            "2009-02-20- 20"; volume: "284"; issue: "8"; pages: "5324-31"
COMMENT     Sequence Label: pRSF- RPA4 (MSW#716)
FEATURES             Location/Qualifiers
     source          1..4568
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     956..975
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(995..1072)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        1118..1136
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1137..1161
                     /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             1217..1234
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        1348..1366
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1367..1391
                     /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             2239..2283
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2321..2339)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2335..2382
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2612..3427)
                     /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(2612..3427)
                     /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     3335..3354
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3428..3519)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3535..4284)
                     /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(3545..3562)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3785..3804)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4480..4568,1..994))
                     /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"
ORIGIN
        1 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
       61 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
      121 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
      181 ccactaccga gatgtccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
      241 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
      301 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
      361 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
      421 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
      481 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
      541 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
      601 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
      661 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
      721 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
      781 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
      841 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
      901 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
      961 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     1021 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     1081 cgacgctctc ccttatgcga ctcctgcatt aggaaattaa tacgactcac tataggggaa
     1141 ttgtgagcgg ataacaattc ccctgtagaa ataattttgt ttaactttaa taaggagata
     1201 taccatgggc agcagccatc accatcatca ccacagccag gatccgaatt cgagctcggc
     1261 gcgcctgcag gtcgacaagc ttgcggccgc ataatgctta agtcgaacag aaagtaatcg
     1321 tattgtacac ggccgcataa tcgaaattaa tacgactcac tataggggaa ttgtgagcgg
     1381 ataacaattc cccatcttag tatattagtt aagtataaga aggagatata cataatgagt
     1441 aagagtgggt ttgggagcta tggcagcatt tctgctgctg atggagcgag tggaggcagt
     1501 gaccaactgt gtgagagaga tgcaactcct gctattaaga cccaaagacc taaggtccga
     1561 attcaggacg ttgtaccgtg taacgtgaac cagctactca gctctactgt gtttgaccct
     1621 gtgttcaagg ttaggggaat tatagtttcc caggtctcca tcgtgggggt aatcagaggg
     1681 gcagagaagg cttcaaatca catttgttac aaaattgatg atatgaccgc gaaaccaatc
     1741 gaggcccgac agtggtttgg tagagagaaa gtcaagcagg tgactccatt gtcagtcgga
     1801 gtatatgtca aagtgtttgg tatcctcaaa tgtcccacgg gaacaaagag ccttgaggta
     1861 ttgaaaattc atgtcctaga ggacatgaac gagttcaccg tgcatattct ggaaacggtc
     1921 aatgcacaca tgatgctgga taaagcccgt cgtgatacca ctgtagaaag tgtgcctgtg
     1981 tctccatcag aagtgaatga tgctggggat aacgatgaga gtcaccgcaa tttcatccag
     2041 gacgaagtgc tgcgtttgat tcatgagtgt cctcatcagg aagggaagag catccatgag
     2101 ctccgggctc agctctgcga ccttagcgtc aaggccatca aggaagcgat tgattatctg
     2161 accgttgagg gccacatcta tcccactgtg gatcgggagc attttaagtc tgctgattga
     2221 ggtaccctcg agtctggtaa agaaaccgct gctgcgaaat ttgaacgcca gcacatggac
     2281 tcgtctacta gcgcagctta attaacctag gctgctgcca ccgctgagca ataactagca
     2341 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaacc tcaggcattt
     2401 gagaagcaca cggtcacact gcttccggta gtcaataaac cggtaaacca gcaatagaca
     2461 taagcggcta tttaacgacc ctgccctgaa ccgacgacaa gctgacgacc gggtctccgc
     2521 aagtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2581 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     2641 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     2701 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     2761 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     2821 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     2881 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     2941 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcggtcgc
     3001 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     3061 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     3121 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     3181 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     3241 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     3301 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     3361 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     3421 ggctcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     3481 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggcatgcag cgctcttccg
     3541 cttcctcgct cactgactcg ctacgctcgg tcgttcgact gcggcgagcg gtgtcagctc
     3601 actcaaaagc ggtaatacgg ttatccacag aatcagggga taaagccgga aagaacatgt
     3661 gagcaaaaag caaagcaccg gaagaagcca acgccgcagg cgtttttcca taggctccgc
     3721 ccccctgacg agcatcacaa aaatcgacgc tcaagccaga ggtggcgaaa cccgacagga
     3781 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     3841 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     3901 agctcacgct gttggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     3961 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4021 aacccggtaa gacacgactt atcgccactg gcagcagcca ttggtaactg atttagagga
     4081 ctttgtcttg aagttatgca cctgttaagg ctaaactgaa agaacagatt ttggtgagtg
     4141 cggtcctcca acccacttac cttggttcaa agagttggta gctcagcgaa ccttgagaaa
     4201 accaccgttg gtagcggtgg tttttcttta tttatgagat gatgaatcaa tcggtctatc
     4261 aagtcaacga acagctattc cgttactcta gatttcagtg caatttatct cttcaaatgt
     4321 agcacctgaa gtcagcccca tacgatataa gttgtaattc tcatgttagt catgccccgc
     4381 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     4441 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     4501 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     4561 cgtattgg
//
