LOCUS       sequence-203021-        5658 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  in vitro transcription, cell cycle nuclear marker .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5658)
  AUTHORS   Daniel K, Icha J, Horenburg C, Muller D, Norden C, Mansfeld J
  TITLE     Conditional control of fluorescent protein degradation by an 
            auxin-dependent nanobody.
  JOURNAL   Nat Commun. 2018 Aug 17;9(1):3297. doi: 10.1038/s41467-018-05855-5.
  PUBMED    30120238
REFERENCE   2  (bases 1 to 5658)
  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"; journalName: "Nat 
            Commun."; date: "2018-08-17"; volume: "9(1):3297. doi"; pages: " 
            10.1038/s41467-018-05855-5"
COMMENT     Sequence Label: pCS2 PCNA GFP
FEATURES             Location/Qualifiers
     source          1..5658
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             85..105
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      118..127
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(124..126,127..129,130..840)
                     /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(169..190)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(430..449)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     777..798
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1710..1844
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1734..1753)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1788..1807
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1957..1977)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1957..1975)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1996..2012)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1996..2012)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2009..2031)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2020..2036
                     /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(2044..2050,2051..2068,2069..2074))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2089..2110
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2227..2244)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2398..2986)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2478..2497)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3157..3948,3949..4017))
                     /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     3780..3799
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4018..4122)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4148..4603)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4285..4306)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4497..4516
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4674..5658
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atccccgaag aagaagcgca aagtactggt accggtcgcc
      121 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
      181 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
      241 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
      301 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
      361 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
      421 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
      481 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
      541 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
      601 aacggcatca aggccaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
      661 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
      721 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
      781 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
      841 ggcgaagggc aagggcaagg gcaagggccg ggccgcggct acgcgtatcg atccatgttc
      901 gaggcgcgcc tggtccaggg ctccatcctc aagaaggtgt tggaggcact caaggacctc
      961 atcaacgagg cctgctggga tattagctcc agcggtgtaa acctgcagag catggactcg
     1021 tcccacgtct ctttggtgca gctcaccctg cggtctgagg gcttcgacac ctaccgctgc
     1081 gaccgcaacc tggccatggg cgtgaacctc accagtatgt ccaaaatact aaaatgcgcc
     1141 ggcaatgaag atatcattac actaagggcc gaagataacg cggatacctt ggcgctagta
     1201 tttgaagcac caaaccagga gaaagtttca gactatgaaa tgaagttgat ggatttagat
     1261 gttgaacaac ttggaattcc agaacaggag tacagctgtg tagtaaagat gccttctggt
     1321 gaatttgcac gtatatgccg agatctcagc catattggag atgctgttgt aatttcctgt
     1381 gcaaaagacg gagtgaaatt ttctgcaagt ggagaacttg gaaatggaaa cattaaattg
     1441 tcacagacaa gtaatgtcga taaagaggag gaagctgtta ccatagagat gaatgaacca
     1501 gttcaactaa cttttgcact gaggtacctg aacttcttta caaaagccac tccactctct
     1561 tcaacggtga cactcagtat gtctgcagat gtaccccttg ttgtagagta taaaattgcg
     1621 gatatgggac acttaaaata ctacttggct cccaagatcg aggatgaaga aggatcttag
     1681 tctagaacta tagtgagtcg tattacgtag atccagacat gataagatac attgatgagt
     1741 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     1801 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
     1861 ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaattc gcggccgcgg
     1921 cgccaatgca ttgggcccgg tacccagctt ttgttccctt tagtgagggt taattgcgcg
     1981 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     2041 acacaacata cgagccggga gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     2101 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     2161 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     2221 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     2281 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     2341 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     2401 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     2461 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     2521 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     2581 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     2641 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     2701 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     2761 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     2821 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     2881 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     2941 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     3001 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     3061 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     3121 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     3181 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     3241 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     3301 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     3361 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     3421 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     3481 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     3541 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     3601 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     3661 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     3721 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     3781 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     3841 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     3901 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     3961 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     4021 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     4081 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     4141 gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     4201 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     4261 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     4321 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     4381 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     4441 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     4501 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     4561 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg
     4621 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagtcgacc
     4681 gccaattcaa tatggcgtat atggactcat gccaattcaa tatggtggat ctggacctgt
     4741 gccaattcaa tatggcgtat atggactcgt gccaattcaa tatggtggat ctggacccca
     4801 gccaattcaa tatggcggac ttggcaccat gccaattcaa tatggcggac ctggcactgt
     4861 gccaactggg gaggggtcta cttggcacgg tgccaagttt gaggaggggt cttggccctg
     4921 tgccaagtcc gccatattga attggcatgg tgccaataat ggcggccata ttggctatat
     4981 gccaggatca atatataggc aatatccaat atggccctat gccaatatgg ctattggcca
     5041 ggttcaatac tatgtattgg ccctatgcca tatagtattc catatatggg ttttcctatt
     5101 gacgtagata gcccctccca atgggcggtc ccatatacca tatatggggc ttcctaatac
     5161 cgcccatagc cactccccca ttgacgtcaa tggtctctat atatggtctt tcctattgac
     5221 gtcatatggg cggtcctatt gacgtatatg gcgcctcccc cattgacgtc aattacggta
     5281 aatggcccgc ctggctcaat gcccattgac gtcaatagga ccacccacca ttgacgtcaa
     5341 tgggatggct cattgcccat tcatatccgt tctcacgccc cctattgacg tcaatgacgg
     5401 taaatggccc acttggcagt acatcaatat ctattaatag taacttggca agtacattac
     5461 tattggaagt acgccagggt acattggcag tactcccatt gacgtcaatg gcggtaaatg
     5521 gcccgcgatg gctgccaagt acatccccat tgacgtcaat ggggaggggc aatgacgcaa
     5581 atgggcgttc cattgacgta aatgggcggt aggcgtgcct aatgggaggt ctatataagc
     5641 aatgctcgtt tagggaac
//
