LOCUS       sequence-251684-        5078 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains insert allowing in-vivo visualization of Cdk1 protein (G1).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5078)
  AUTHORS   Tran M, Dave M, Lansford R
  TITLE     Fast and Efficient Expression of Multiple Proteins in Avian Embryos 
            Using mRNA Electroporation.
  JOURNAL   J Vis Exp. 2019 Jun 7;(148). doi: 10.3791/59664.
  PUBMED    31233018
REFERENCE   2  (bases 1 to 5078)
  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: "J Vis 
            Exp."; date: "2019-06-7"; volume: "(148). doi"; pages: " 
            10.3791/59664"
COMMENT     Sequence Label: pCS2+FucciOrange
FEATURES             Location/Qualifiers
     source          1..5078
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..978
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        1013..1031
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1013..1030
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      1083..1092
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1089..1739
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAH"
     CDS             1794..2066
                     /codon_start=1
                     /product="degron consisting of residues 30-120 of human 
                     Cdt1 (Zielke and Edgar, 2015)"
                     /label=Cdt1 (30-120)
                     /translation="PSPARPALRAPASATSGSRKRARPPAAPGRDQARPPARRRLRLSV
                     DEVSSPSTPEAPDIPACPSPGQKIKKSTPAAGQPPHLTSAQDQDTI"
     polyA_signal    2108..2242
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2132..2151)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2186..2205
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2355..2375)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2355..2373)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2394..2410)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2394..2410)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2407..2429)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2418..2434
                     /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(2442..2448,2449..2466,2467..2472))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2487..2508
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2625..2642)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2796..3384)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2876..2895)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3555..4346,4347..4415))
                     /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     4178..4197
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4416..4520)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4546..5001)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4683..4704)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4895..4914
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gccaattcaa tatggcgtat atggactcat gccaattcaa tatggtggat ctggacctgt
       61 gccaattcaa tatggcgtat atggactcgt gccaattcaa tatggtggat ctggacccca
      121 gccaattcaa tatggcggac ttggcaccat gccaattcaa tatggcggac ctggcactgt
      181 gccaactggg gaggggtcta cttggcacgg tgccaagttt gaggaggggt cttggccctg
      241 tgccaagtcc gccatattga attggcatgg tgccaataat ggcggccata ttggctatat
      301 gccaggatca atatataggc aatatccaat atggccctat gccaatatgg ctattggcca
      361 ggttcaatac tatgtattgg ccctatgcca tatagtattc catatatggg ttttcctatt
      421 gacgtagata gcccctccca atgggcggtc ccatatacca tatatggggc ttcctaatac
      481 cgcccatagc cactccccca ttgacgtcaa tggtctctat atatggtctt tcctattgac
      541 gtcatatggg cggtcctatt gacgtatatg gcgcctcccc cattgacgtc aattacggta
      601 aatggcccgc ctggctcaat gcccattgac gtcaatagga ccacccacca ttgacgtcaa
      661 tgggatggct cattgcccat tcatatccgt tctcacgccc cctattgacg tcaatgacgg
      721 taaatggccc acttggcagt acatcaatat ctattaatag taacttggca agtacattac
      781 tattggaagt acgccagggt acattggcag tactcccatt gacgtcaatg gcggtaaatg
      841 gcccgcgatg gctgccaagt acatccccat tgacgtcaat ggggaggggc aatgacgcaa
      901 atgggcgttc cattgacgta aatgggcggt aggcgtgcct aatgggaggt ctatataagc
      961 aatgctcgtt tagggaaccg ccattctgcc tggggacgtc ggagcaagct tgatttaggt
     1021 gacactatag aatacaagct acttgttctt tttgcaggat cccatcgatt cgaattctgc
     1081 aggccaccat ggtgagtgtg attaaaccag agatgaagat gaggtactac atggacggct
     1141 ccgtcaatgg gcatgagttc acaattgaag gtgaaggcac aggcagacct tacgagggac
     1201 atcaagagat gacactacgc gtcacaatgg ccgagggcgg gccaatgcct ttcgcgtttg
     1261 acttagtgtc acacgtgttc tgttacggcc acagagtatt tactaaatat ccagaagaga
     1321 taccagacta tttcaaacaa gcatttcctg aaggcctgtc atgggaaagg tcgttggagt
     1381 tcgaagatgg tgggtccgct tcagtcagtg cgcatataag ccttagagga aacaccttct
     1441 accacaaatc caaatttact ggggttaact ttcctgccga tggtcctatc atgcaaaacc
     1501 aaagtgttga ttgggagcca tcaaccgaga aaattactgc cagcgacgga gttctgaagg
     1561 gtgatgttac gatgtaccta aaacttgaag gaggcggcaa tcacaaatgc caaatgaaga
     1621 ctacttacaa ggcggcaaaa gagattcttg aaatgccagg agaccattac atcggccatc
     1681 gcctcgtcag gaaaaccgaa ggcaacatta ctgagcaggt agaagatgca gtagctcatt
     1741 actcaatgtt gccttcccag ggcggatatc catcacactg gcggccgctc gagcccagcc
     1801 ccgccaggcc cgcactccgc gccccggcct ccgctaccag tggcagccgc aagcgcgccc
     1861 gcccgcccgc cgcccccgga cgcgaccagg ccaggccacc ggcccgcagg agactgcggc
     1921 tgtcggtgga cgaggtttcc agccccagta cccccgaggc cccagacatc ccagcctgcc
     1981 cttctccggg ccagaagata aagaaatcca ccccggcagc aggtcagccg ccccacctga
     2041 catccgcgca ggaccaggac accatcggat ctcgagcctc tagaactata gtgagtcgta
     2101 ttacgtagat ccagacatga taagatacat tgatgagttt ggacaaacca caactagaat
     2161 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     2221 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     2281 gggggaggtg tgggaggttt tttaattcgc ggccgcggcg ccaatgcatt gggcccggta
     2341 cccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag
     2401 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccgggagc
     2461 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     2521 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     2581 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     2641 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     2701 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     2761 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     2821 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     2881 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     2941 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     3001 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     3061 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     3121 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     3181 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     3241 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     3301 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     3361 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     3421 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     3481 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     3541 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     3601 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     3661 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     3721 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     3781 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     3841 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     3901 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     3961 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     4021 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     4081 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     4141 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     4201 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     4261 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     4321 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     4381 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     4441 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     4501 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta
     4561 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     4621 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     4681 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     4741 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     4801 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     4861 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     4921 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     4981 atgcgccgct acagggcgcg tcccattcgc cattcaggct gcgcaactgt tgggaagggc
     5041 gatcggtgcg ggcctcttcg ctattacgcc agtcgacc
//
