LOCUS       sequence-251683-        5132 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains insert allowing in-vivo visualization of CyclinB1 protein 
            (G2/M).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5132)
  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 5132)
  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+CyclinB1-GFP
FEATURES             Location/Qualifiers
     source          1..5132
                     /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"
     CDS             join(1405..1407,1408..1410,1411..2124)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1405..1407,1408..1410,1411..2121)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1450..1471)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1711..1730)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2058..2079
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2162..2296
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2186..2205)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2240..2259
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2409..2429)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2409..2427)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2448..2464)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2448..2464)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2461..2483)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2472..2488
                     /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(2496..2502,2503..2520,2521..2526))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2541..2562
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2679..2696)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2850..3438)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2930..2949)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3609..4400,4401..4469))
                     /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     4232..4251
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4470..4574)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4600..5055)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4737..4758)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4949..4968
                     /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 ccaccatggc gctcagggtc
     1081 actaggaaca cgaaaattaa cgcagaaaat aaggccaagg tcagtatggc aggcgccaag
     1141 cgtgtgcctg tgacagttac tgctgcttcc aagcccgggc tgagaccgag aactgctctt
     1201 ggagacattg gtaataaagt cagcgaagag ctacaggcaa gagtgcctct gaaaagggaa
     1261 gcaaaaacgc taggtactgg aaaaggtact gttaaagccc taccaaaacc tgtagagaag
     1321 gtgcctgtgt gtgaaccaga ggtggaactt gctgagcctg agcctgaacc tgaacttgaa
     1381 ctagttcaga atttgtctag agccatggtg agcaagggcg aggagctgtt caccggggtg
     1441 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     1501 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     1561 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     1621 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     1681 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     1741 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     1801 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     1861 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     1921 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     1981 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     2041 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     2101 ggcatggacg agctgtacaa gtaagtcgag cctctagaac tatagtgagt cgtattacgt
     2161 agatccagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg
     2221 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     2281 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga
     2341 ggtgtgggag gttttttaat tcgcggccgc ggcgccaatg cattgggccc ggtacccagc
     2401 ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt
     2461 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg gagcataaag
     2521 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     2581 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     2641 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     2701 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     2761 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     2821 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     2881 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     2941 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     3001 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     3061 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     3121 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     3181 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     3241 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     3301 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     3361 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     3421 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     3481 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     3541 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     3601 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     3661 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     3721 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     3781 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     3841 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     3901 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     3961 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     4021 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     4081 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     4141 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     4201 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     4261 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     4321 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     4381 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     4441 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     4501 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     4561 ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt
     4621 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     4681 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     4741 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     4801 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     4861 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     4921 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     4981 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     5041 cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg
     5101 tgcgggcctc ttcgctatta cgccagtcga cc
//
