LOCUS       sequence-240735-        7656 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of GFP-Dub3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7656)
  AUTHORS   van_der_Laan S, Tsanov N, Crozet C, Maiorano D
  TITLE     High Dub3 expression in mouse ESCs couples the G1/S checkpoint to 
            pluripotency.
  JOURNAL   Mol Cell. 2013 Nov 7;52(3):366-79. doi: 
            10.1016/j.molcel.2013.10.003.
  PUBMED    24207026
REFERENCE   2  (bases 1 to 7656)
  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.2013.10"; journalName: "Mol Cell"; date: 
            "2013-11-7- 7"; volume: "52"; issue: "3"; pages: "366-79"
COMMENT     Sequence Label: pcDNA3-GFP-Dub3 mouse
FEATURES             Location/Qualifiers
     source          1..7656
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      671..680
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(677..679,680..682,683..1393)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(677..679,680..682,683..1393)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(722..743)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(983..1002)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1330..1351
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     RBS             2654..2662
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     promoter        complement(2975..2993)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2976..2993)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3013..3030)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3019..3243
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3289..3717
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3376..3395)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3586..3607
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3726..3746)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3731..4060
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3911..4046
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3973..3992
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4127..4921
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4181..4200)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4791..4810
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5095..5216
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5132..5151)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5186..5205
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5265..5281)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5265..5281)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5278..5300)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5289..5305
                     /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(5313..5319,5320..5337,5338..5343))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5358..5379
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5496..5513)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5667..6255)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5747..5766)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6426..7217,7218..7286))
                     /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     7049..7068
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7287..7391)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7466..7485)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 ggtaccggtc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
      721 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
      781 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
      841 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
      901 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
      961 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     1021 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     1081 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     1141 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     1201 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     1261 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     1321 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     1381 cgagctgtac aagtccggcc ggactcagat ctcgagctca agcttcgagg ggaattctat
     1441 ggtggttgct ctttccttcc cagaagcaga tccagcacta tcatctcctg atgccccaga
     1501 gctgcatcag gatgaagctc aggtggtgga ggagctaact accaatggaa agcacagtct
     1561 gagttgggag agtccccaag gaccaggatg cgggctccag aacacaggca acagctgcta
     1621 cctgaacgca gccctgcagt gcttgacaca cacaccacct ctagctgact acatgctgtc
     1681 ccaggagcac agtcaaacct gttgttcccc agaaggctgt aagatgtgtg ctatggaagc
     1741 ccatgtgacc cagagtctcc tgcactctca ctcgggggat gtcatgaagc cctcccagat
     1801 tttgacctct gccttccaca agcaccagca ggaagatgcc catgagtttc tcatgttcac
     1861 cttggaaaca atgcatgaat cctgccttca agtgcacaga caatcagatc ccacccctca
     1921 ggatacgtca cccattcatg acatatttgg aggctggtgg aggtctcaga tcaagtgtct
     1981 ccattgccag ggcacctcac ataccttcga tcccttcctg gatgtccccc tggatatcag
     2041 ctcagctcag agtgtaaatc aagccttgtg ggatacaggg aagtcagaag agctacttgg
     2101 agagaatgcc tactactgtg gtaggtgtag acagaagatg ccagcttcta agaccctgca
     2161 tgttcatatt gctccaaagg tactcctgct agtgttaaag cgcttctcag ccttcacggg
     2221 taacaagtta gacagaaaag taagctaccc ggagttcctt gacctgaagc catacctgtc
     2281 tgagcctact ggaggacctt tgccttatgc cctctatgcc gtcctggtcc atgatggtgc
     2341 gacttctaac agtggacatt acttctgttg tgtcaaagct ggtcatggga agtggtacaa
     2401 gatggatgat actaaggtca ccaggtgtga tgtgacttct gtcctgaatg agaatgccta
     2461 tgtgctcttc tatgtgcagc agaccgacct caaacaggtc agtattgaca tgccagaggg
     2521 cagagtacat gaggttcttg accctaaata ccagctgaag aaatcccgga gaaaaaagcg
     2581 taagaagcaa tgccattgca cagatgatgc cggagaggca tgcgaaaaca gggagaagag
     2641 agcaaagaaa gaaacctcct taggggaggg gaaagtgcct caggaagtga accacgagaa
     2701 agctgggcag aaacatggga ataccaaact cgtgcctcag gaacagaacc accagagagc
     2761 tgggcagaac ctcaggaata ctgaagttga acttgatctg cctgttgatg caattgtgat
     2821 tcaccagccc agatccacag caaactgggg cacggatgct ccagacaaag agaatcaacc
     2881 ctggcacaat ggtgacaggc tcctcacctc tcagggcctc atgagccctg ggcagctctg
     2941 tagtcagggt gggagataat ctagagggcc ctattctata gtgtcaccta aatgctagag
     3001 ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     3061 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     3121 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     3181 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     3241 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     3301 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     3361 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     3421 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     3481 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     3541 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     3601 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     3661 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     3721 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     3781 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     3841 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     3901 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     3961 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
     4021 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
     4081 atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     4141 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     4201 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     4261 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
     4321 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     4381 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     4441 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     4501 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     4561 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     4621 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt
     4681 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     4741 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     4801 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     4861 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     4921 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     4981 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     5041 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     5101 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     5161 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     5221 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     5281 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     5341 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     5401 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     5461 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     5521 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     5581 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     5641 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     5701 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5761 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5821 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5881 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5941 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     6001 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     6061 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     6121 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     6181 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     6241 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     6301 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     6361 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     6421 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     6481 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     6541 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     6601 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     6661 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     6721 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6781 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6841 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6901 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6961 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     7021 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     7081 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     7141 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     7201 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     7261 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     7321 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     7381 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc
     7441 ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     7501 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta
     7561 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg
     7621 cgctgcttcg cgatgtacgg gccagatata cgcgtt
//
