LOCUS       sequence-386845-        8118 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mRFP tagged human PCNA in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8118)
  AUTHORS   Heinz KS, Casas-Delucchi CS, Torok T, Cmarko D, Rapp A, Raska I, 
            Cardoso MC
  TITLE     Peripheral re-localization of constitutive heterochromatin advances 
            its replication timing and impairs maintenance of silencing marks.
  JOURNAL   Nucleic Acids Res. 2018 Jul 6;46(12):6112-6128. doi: 
            10.1093/nar/gky368.
  PUBMED    29750270
REFERENCE   2  (bases 1 to 8118)
  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"; journalName: 
            "Nucleic Acids Res"; date: "2018-07-6- 6"; volume: "46"; issue: 
            "12"; pages: "6112-6128"
COMMENT     Sequence Label: pEN-CAG-mRFP-PCNA pc2729
FEATURES             Location/Qualifiers
     source          1..8118
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    578..712
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        726..1105
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1107..1382
                     /label=chicken beta-actin promoter
     intron          1383..2399
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2323..2345
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2407..2426
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     5'UTR           2460..2499
                     /label=HSV TK 5'-UTR
                     /note="5' untranslated region from the herpes simplex virus
                     thymidine kinase gene"
     CDS             2535..2555
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2571..3245
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             2571..3245
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(2701..2719)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2969..2988
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3163..3186
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     misc_feature    4216..4768
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4360..4377)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4587..4606
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4780..5379
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4780..4799)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5276..5296
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5460..5477)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5466..5690
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5737..5753)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5737..5753)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5750..5772)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5761..5777
                     /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)."
     promoter        complement(join(5785..5791,5792..5809,5810..5815))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5830..5851
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5910..6105
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5956..6091
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6111..6245
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6313..6330)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6484..7072)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6564..6583)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7243..8034,8035..8103))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7866..7885
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 atgaccgctt tggaaaaaca aagactgtat ttcctggaaa ttaatgttta ttcaataaac
       61 tgtgtattca gctatattca catagtggtg aggctgaaat gaggcgggaa gaggcggttg
      121 gggcttaatt atatcaattt gggtggcccc acagcgcctc caaggcgcca gtcctgtttt
      181 gacaagttgc ctctggaagc ctctcttctt tttctccaga gtaagcggag gccaggggcc
      241 cccggcctct gcttaatact aaaaaaaaca gctgttgtca tagtaatgat tgggtggaaa
      301 cattccaggc ctgggtggag aggctttttg cttcctcttg caaaaccaca ctgacattcc
      361 aggcctgggt ggagaggctt tttgcttcct cttgcaaaac cacactgccc tctggagggc
      421 agttgcctag caactaatta aaagaggatg tcgcacggcc agctgcggtc agttagtcac
      481 ttcctgctta actgacttga cattttctat tttaagagtc gggaggaaaa ttactgtgtt
      541 ggaggccctc cgccatcttc tgaagctgaa tcgaattaac ttgtttattg cagcttataa
      601 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
      661 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga tctgatatca
      721 tcgtcgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca
      781 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
      841 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
      901 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
      961 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
     1021 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
     1081 cgtattagtc atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc
     1141 catctccccc ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc
     1201 agcgatgggg gcgggggggg ggggggggsg cgccrggsgg ggcggggsgg ggcgaggggc
     1261 ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt
     1321 ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg
     1381 cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc
     1441 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
     1501 ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg tggctgcgtg
     1561 aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg gggggtgcgt
     1621 gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc ggctgtgagc
     1681 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg agcgcggccg
     1741 ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa caaaggctgc gtgcggggtg
     1801 tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt cgggctgcaa ccccccctgc
     1861 acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtacggggc
     1921 gtggcgcggg gctcgccgtg ccgggcgggg ggkggcggca ggkgggggkg ccgggcgggg
     1981 cggggccgcc kcgggccggg gggggckcgg gggrggggcg cggcggcccc cggagcgccg
     2041 gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg tgcgagaggg
     2101 cgcagggact tcctttgtcc caaatctgtg cggagccgaa atctgggagg cgccgccgca
     2161 ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg
     2221 agggccttcg tgcgtcgccg cgccgccgtc cccttctccc tctccagcct cggggctgtc
     2281 cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg
     2341 tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc
     2401 tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa ttcctcgagg
     2461 atcttggtgg cgtgaaactc ccgcacctct tcggccagcg ccttgtagaa gcgcgtatgg
     2521 cttcgtgggg atccccgaag aagaagcgca aagtactggt accggtcgac atggcctcct
     2581 ccgaggacgt catcaaggag ttcatgcgct tcaaggtgcg catggagggc tccgtgaacg
     2641 gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg
     2701 ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc ctgtcccctc
     2761 agttccagta cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga
     2821 agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg
     2881 tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc
     2941 tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg
     3001 aggcctccac cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagatga
     3061 ggctgaagct gaaggacggc ggccactacg acgccgaggt caagaccacc tacatggcca
     3121 agaagcccgt gcagctgccc ggcgcctaca agaccgacat caagctggac atcacctccc
     3181 acaacgagga ctacaccatc gtggaacagt acgagcgcgc cgagggccgc cactccaccg
     3241 gcgccggcct gtacaagggc gaagggcaag ggcaagggca agggccgggc cgcggctacg
     3301 cgtatcgatc catgttcgag gcgcgcctgg tccagggctc catcctcaag aaggtgttgg
     3361 aggcactcaa ggacctcatc aacgaggcct gctgggatat tagctccagc ggtgtaaacc
     3421 tgcagagcat ggactcgtcc cacgtctctt tggtgcagct caccctgcgg tctgagggct
     3481 tcgacaccta ccgctgcgac cgcaacctgg ccatgggcgt gaacctcacc agtatgtcca
     3541 aaatactaaa atgcgccggc aatgaagata tcattacact aagggccgaa gataacgcgg
     3601 ataccttggc gctagtattt gaagcaccaa accaggagaa agtttcagac tatgaaatga
     3661 agttgatgga tttagatgtt gaacaacttg gaattccaga acaggagtac agctgtgtag
     3721 taaagatgcc ttctggtgaa tttgcacgta tatgccgaga tctcagccat attggagatg
     3781 ctgttgtaat ttcctgtgca aaagacggag tgaaattttc tgcaagtgga gaacttggaa
     3841 atggaaacat taaattgtca cagacaagta atgtcgataa agaggaggaa gctgttacca
     3901 tagagatgaa tgaaccagtt caactaactt ttgcactgag gtacctgaac ttctttacaa
     3961 aagccactcc actctcttca acggtgacac tcagtatgtc tgcagatgta ccccttgttg
     4021 tagagtataa aattgcggat atgggacact taaaatacta cttggctccc aagatcgagg
     4081 atgaagaagg atcttagtct agagctgaga acttcagggt gagtttggct taagtttaaa
     4141 ccgctgatca gcctcgacta cggccgctcg ataagcttga tatcgaattc cgcccccccc
     4201 ccyccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg
     4261 tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg
     4321 gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag
     4381 gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt
     4441 ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc
     4501 aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga
     4561 gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga
     4621 aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct
     4681 ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg
     4741 ttttcctttg aaaaacacga tgataatatg gccacaacca tgaccgagta caagcccacg
     4801 gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc
     4861 gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc
     4921 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg
     4981 gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg
     5041 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa
     5101 cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc
     5161 gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga
     5221 gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac
     5281 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga
     5341 ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgac gcccgcccca cgacccgcag
     5401 cgcccgaccg aaaggagcgc acgaccccat gcatcgatga tctagagctc gctgatcagc
     5461 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     5521 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     5581 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     5641 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     5701 ggaaagaacc tgcagcccaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     5761 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     5821 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     5881 gtcgggaaac ctgtcgtgcc agcggatccg catctcaatt agtcagcaac catagtcccg
     5941 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     6001 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     6061 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct aacttgttta
     6121 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     6181 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     6241 ggatccgctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     6301 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     6361 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     6421 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     6481 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     6541 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     6601 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     6661 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     6721 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     6781 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     6841 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     6901 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     6961 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     7021 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     7081 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     7141 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     7201 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     7261 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     7321 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     7381 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     7441 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     7501 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     7561 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     7621 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     7681 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     7741 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     7801 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     7861 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     7921 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     7981 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     8041 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     8101 catactcttc ctttttca
//
