LOCUS       sequence-380238-        6335 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Human Cdt1 (SV40 NLS localization and C-terminal mCitrine tag) with 
            mutations which abroggate its S phase degradation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6335)
  AUTHORS   Ratnayeke N, Baris Y, Chung M, Yeeles JTP, Meyer T
  TITLE     CDT1 inhibits CMG helicase in early S phase to separate origin 
            licensing from DNA synthesis.
  JOURNAL   Mol Cell. 2023 Jan 5;83(1):26-42.e13. doi: 
            10.1016/j.molcel.2022.12.004.
  PUBMED    36608667
REFERENCE   2  (bases 1 to 6335)
  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.2022.12"; journalName: "Mol Cell"; date: 
            "2023-01-5- 5"; volume: "83"; issue: "1"; pages: "26-42.e13"
COMMENT     Sequence Label: pC1-ND-Cdt1-mCitrine
FEATURES             Location/Qualifiers
     source          1..6335
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     187..206
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(505..524)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    549..596
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      925..1513
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1414..1433
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1688..1991
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1992..2195
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2146..2166
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             3875..3895
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3908..3910,3911..3913,3914..4627)
                     /codon_start=1
                     /product="enhanced YFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3953..3974)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4214..4233)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4561..4582
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4750..4871
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4787..4806)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4841..4860
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4878..5333)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5015..5036)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5227..5246
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5360..5464
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5466..5823
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5466..5657
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5489..5509)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5674..5809
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(5858..6335,1..317)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             join(5858..6335,1..317)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5912..5931)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg agcatgcccg
       61 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
      121 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
      181 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
      241 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
      301 ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa
      361 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
      421 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
      481 cgcccaccct agggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg
      541 cgctatgacg gcaataaaaa gacagaataa aacgcacggt gttgggtcgt ttgttcataa
      601 acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac cccattgggg
      661 ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg gtgaaggccc
      721 agggctcgca gccaacgtcg gggcggcagg ccctgccata gcctcaggtt actcatatat
      781 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
      841 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
      901 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
      961 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     1021 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     1081 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     1141 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     1201 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     1261 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     1321 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     1381 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     1441 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     1501 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     1561 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     1621 catgcattag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
     1681 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
     1741 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
     1801 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
     1861 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
     1921 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
     1981 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
     2041 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
     2101 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
     2161 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
     2221 agcgctaccg gactcagatc tcgagctcaa gcttgccacc atgatagctc cgccaaagct
     2281 cgcttgtcgg actccctctc cggctagacc ggcacttcgc gcccccgcct ccgctacttc
     2341 aggtagccga aagagggcga ggccaccagc agcccccgga cgcgatcagg cgaggccacc
     2401 tgctcgagcg gctgctcgcc tctcagtaga tgaggttagc agccccagta cccccgaggc
     2461 cccagacatc ccagcctgcc cttctccggg ccagaagata aagaaatcca ccccggcagc
     2521 aggtcagccg ccccacctga catccgcgca ggaccaggac accatctctg agcttgcgtc
     2581 atgcctgcaa cgggcccggg agctgggggc aagagtccgg gcgctgaagg ccagtgccca
     2641 ggatgctggg gagtcctgca ccccagaggc cgagggccgc cctgaggagc catgtggcga
     2701 gaaggcgccc gcctaccagc gcttccatgc cctggcccag cccggcctgc cgggactcgt
     2761 gctgccctac aagtaccagg tgctggcgga gatgttccgc agcatggaca ccatcgtggg
     2821 catgctccac aaccgctccg agacgcccac ctttgccaag gtccagcggg gcgtccagga
     2881 catgatgcgt aggcgttttg aggagcgcaa tgttggccag atcaaaaccg tgtacccggc
     2941 ctcctaccgc ttccgccagg agcgcagtgt ccccaccttc aaggatggca ccaggaggtc
     3001 agattaccag ctcaccatcg agccactgct ggagcaggag gctgacggag cagcccccca
     3061 gctcacggcc tcgcgcctcc tgcagcgacg gcagatcttc agccagaagc tggtggagca
     3121 tgtcaaggag caccacaagg ccttcctggc ctccctgagc cccgccatgg tggtgccgga
     3181 ggaccagctg acccgctggc acccgcgctt caacgtggat gaagtacccg acatcgagcc
     3241 ggccgcgctg ccccagccac ccgccacgga gaagctcacc actgctcagg aggtgctggc
     3301 ccgggcccgc aacctgattt cacccaggat ggagaaggcc ttgagtcaat tggccctgcg
     3361 ctctgctgcg cccagcagcc ccgggtctcc caggccagca ctgccggcta ccccaccagc
     3421 caccccgcct gcagcctctc ccagtgctct gaagggggtg tcccaggatc tgctggagcg
     3481 gatccgagcc aaggaggcac agaagcagct ggcacagatg acgcggtgcc cggagcagga
     3541 gcagcggctg cagcgcttag aacggctgcc tgagctggcc cgcgtgctgc ggagcgtctt
     3601 tgtgtccgaa cgcaagcctg cgctcagcat ggaggtggcc tgtgccagga tggtgggcag
     3661 ctgttgtact atcatgagcc ctggggaaat ggagaagcac ctgctgctcc tctccgagct
     3721 gctgccggac tggctcagcc tccaccgcat ccgcaccgac acctacgtca agctggacaa
     3781 ggccgcggac ctcgcccaca tcactgcacg cctggcccac cagacacgtg ctgaggaggg
     3841 gctgggtgca ggaagcgctg ggggatccat ggcacctaag aaaaagcgga aagtgggcgg
     3901 aagtggtatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     3961 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
     4021 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     4081 gcccaccctc gtgaccacct tcggctacgg cctgatgtgc ttcgcccgct accccgacca
     4141 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     4201 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     4261 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     4321 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     4381 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     4441 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     4501 caaccactac ctgagctacc agtccaagct gagcaaagac cccaacgaga agcgcgatca
     4561 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     4621 caagtaaagc ggccgcgact ctagatcata atcagccata ccacatttgt agaggtttta
     4681 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
     4741 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4801 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     4861 aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     4921 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     4981 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     5041 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     5101 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     5161 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     5221 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     5281 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg
     5341 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     5401 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     5461 gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     5521 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     5581 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     5641 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     5701 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     5761 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     5821 aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     5881 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     5941 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     6001 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc
     6061 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     6121 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     6181 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     6241 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     6301 ggaagccggt cttgtcgatc aggatgatct ggacg
//
