LOCUS       sequence-388295-        6025 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The GFP-PCNAL2 fusion protein contains a SV40 nuclear localization 
            signal at the NH2 terminus followed by an enhanced mutant GFP gene 
            that is fused to the human PCNA by a linker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6025)
  AUTHORS   Leonhardt H, Rahn HP, Weinzierl P, Sporbert A, Cremer T, Zink D, 
            Cardoso MC
  TITLE     Dynamics of DNA replication factories in living cells.
  JOURNAL   J Cell Biol. 2000 Apr 17;149(2):271-80. doi: 10.1083/jcb.149.2.271.
  PUBMED    10769021
REFERENCE   2  (bases 1 to 6025)
  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: "10.1083/jcb.149.2";
            journalName: "J Cell Biol"; date: "2000-04-17- 17"; volume: "149"; 
            issue: "2"; pages: "271-80"
COMMENT     Sequence Label: pENeGFP PCNAL2 pc653
FEATURES             Location/Qualifiers
     source          1..6025
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..40
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        21..39
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     enhancer        63..366
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        367..570
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     521..541
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     567..591
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     5'UTR           660..699
                     /label=HSV TK 5'-UTR
                     /note="5' untranslated region from the herpes simplex virus
                     thymidine kinase gene"
     CDS             735..755
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      768..777
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(774..776,777..779,780..1490)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(819..840)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1080..1099)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1427..1448
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     intron          2352..2924
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     primer_bind     complement(3056..3075)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3121..3176
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3175..3194)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     rep_origin      3526..3661
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3767..3784)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3938..4526)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4018..4037)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4697..5488,5489..5557))
                     /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     5320..5339
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5558..5662)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5730..5748
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5786..5808)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5908..5927
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atggtctggc ttatcgaaat taatacgact cactataggg agaccggaat tcgagctcgc
       61 cccgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      121 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      181 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      241 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      301 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      361 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      421 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      481 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      541 gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagat cgcctggaga
      601 cgccatccac gctgttttga cctccataga agacaccggg accgatccag cctccggggg
      661 atcttggtgg cgtgaaactc ccgcacctct tcggccagcg ccttgtagaa gcgcgtatgg
      721 cttcgtgggg atccccgaag aagaagcgca aagtactggt accggtcgcc accatggtga
      781 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
      841 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
      901 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
      961 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
     1021 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     1081 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     1141 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     1201 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
     1261 aggccaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     1321 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     1381 gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     1441 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag ggcgaagggc
     1501 aagggcaagg gcaagggccg ggccgcggct acgcgtatcg atccatgttc gaggcgcgcc
     1561 tggtccaggg ctccatcctc aagaaggtgt tggaggcact caaggacctc atcaacgagg
     1621 cctgctggga tattagctcc agcggtgtaa acctgcagag catggactcg tcccacgtct
     1681 ctttggtgca gctcaccctg cggtctgagg gcttcgacac ctaccgctgc gaccgcaacc
     1741 tggccatggg cgtgaacctc accagtatgt ccaaaatact aaaatgcgcc ggcaatgaag
     1801 atatcattac actaagggcc gaagataacg cggatacctt ggcgctagta tttgaagcac
     1861 caaaccagga gaaagtttca gactatgaaa tgaagttgat ggatttagat gttgaacaac
     1921 ttggaattcc agaacaggag tacagctgtg tagtaaagat gccttctggt gaatttgcac
     1981 gtatatgccg agatctcagc catattggag atgctgttgt aatttcctgt gcaaaagacg
     2041 gagtgaaatt ttctgcaagt ggagaacttg gaaatggaaa cattaaattg tcacagacaa
     2101 gtaatgtcga taaagaggag gaagctgtta ccatagagat gaatgaacca gttcaactaa
     2161 cttttgcact gaggtacctg aacttcttta caaaagccac tccactctct tcaacggtga
     2221 cactcagtat gtctgcagat gtaccccttg ttgtagagta taaaattgcg gatatgggac
     2281 acttaaaata ctacttggct cccaagatcg aggatgaaga aggatcttag tctagagctg
     2341 agaacttcag ggtgagtttg gggacccttg attgttcttt ctttttcgct attgtaaaat
     2401 tcatgttata tggagggggc aaagttttca gggtgttgtt tagaatggga agatgtccct
     2461 tgtatcacca tggaccctca tgataatttt gtttctttca ctttctactc tgttgacaac
     2521 cattgtctcc tcttattttc ttttcatttt ctgtaacttt ttcgttaaac tttagcttgc
     2581 atttgtaacg aatttttaaa ttcacttttg tttatttgtc agattgtaag tactttctct
     2641 aatcactttt ttttcaaggc aatcagggta tattatattg tacttcagca cagttttaga
     2701 gaacaattgt tataattaaa tgataaggta gaatatttct gcatataaat tctggctggc
     2761 gtggaaatat tcttattggt agaaacaact acaccctggt catcatcctg cctttctctt
     2821 tatggttaca atgatataca ctgtttgaga tgaggataaa atactctgag tccaaaccgg
     2881 gcccctctgc taaccatgtt catgccttct tctctttcct acagctcctg ggcaacgtgc
     2941 tggttgttgt gctgtctcat cattttggca aagaattcac tcctcaggtg caggctgcct
     3001 atcagaaggt ggtggctggt gtggccaatg ccctggctca caaataccac tgagatcttt
     3061 ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg acttctggct
     3121 aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt ctctcactcg
     3181 gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg gtttagagtt
     3241 tggcaacata tgcccatatg ctggctgcca tgaacaaagg ttggctataa agaggtcatc
     3301 agtatatgaa acagccccct gctgtccatt ccttattcca tagaaaagcc ttgacttgag
     3361 gttagatttt ttttatattt tgttttgtgt tatttttttc tttaacatcc ctaaaatttt
     3421 ccttacatgt tttactagcc agatttttcc tcctctcctg actactccca gtcatagctg
     3481 tccctcttct cttatggaga tcccggagct ttttgcaaaa gcctaggcct ccaaaaaagc
     3541 ctcttcacta cttctggaat agctcagagg ccgaggcggc ctcggcctct gcataaataa
     3601 aaaaaattag tcagccatgg ggcggagaat gggcggaact gggcggagtt aggggcggga
     3661 tgggcggagt taggggcggg actatggttg ctgactaatt gctgcattaa tgaatcggcc
     3721 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     3781 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     3841 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     3901 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     3961 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     4021 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4081 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     4141 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     4201 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     4261 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     4321 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     4381 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     4441 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     4501 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     4561 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     4621 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     4681 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     4741 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     4801 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     4861 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     4921 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     4981 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     5041 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     5101 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     5161 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     5221 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     5281 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     5341 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     5401 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     5461 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     5521 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     5581 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     5641 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     5701 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg
     5761 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     5821 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     5881 gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     5941 attcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     6001 ccgttgagca ccgccgccgc aagga
//
