LOCUS       sequence-129831-        4943 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GFP reporter protein fused to three mutated GSK3 phosphorylation 
            sites .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4943)
  AUTHORS   Taelman VF, Dobrowolski R, Plouhinec JL, Fuentealba LC, Vorwald PP, 
            Gumper I, Sabatini DD, De Robertis EM
  TITLE     Wnt signaling requires sequestration of glycogen synthase kinase 3 
            inside multivesicular endosomes.
  JOURNAL   Cell. 2010 Dec 23. 143(7):1136-48.
  PUBMED    21183076
REFERENCE   2  (bases 1 to 4943)
  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: "Cell. 2010 
            Dec 23. 143(7):1136-48."
COMMENT     Sequence Label: pCS2 GFP-GSK3mut-MAPK
FEATURES             Location/Qualifiers
     source          1..4943
                     /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"
     regulatory      85..94
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             94..117
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             join(118..120,121..831)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(118..120,121..831)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(160..181)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(421..440)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     768..789
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             931..954
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    995..1129
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1019..1038)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1073..1092
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1242..1262)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1242..1260)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1281..1297)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1281..1297)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1294..1316)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1305..1321
                     /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(1329..1335,1336..1353,1354..1359))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1374..1395
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1512..1529)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1683..2271)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1763..1782)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2442..3233,3234..3302))
                     /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     3065..3084
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3303..3407)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3433..3888)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3570..3591)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3782..3801
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3959..4943
                     /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 atccgccacc atggattaca aggatgacga cgataaggtg
      121 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
      181 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
      241 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
      301 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
      361 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
      421 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
      481 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
      541 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
      601 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
      661 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
      721 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
      781 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gaaatcgaat
      841 tcagctgccg ctgccgctgc cgctgccgct gccgctgccg ctcctgcttc tcctgctgcc
      901 gctgccgctg ccgctgcccc tcctgcttac gactacaagg acgacgatga caagtaactc
      961 gagcctctag aactatagtg agtcgtatta cgtagatcca gacatgataa gatacattga
     1021 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     1081 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
     1141 ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aattcgcggc
     1201 cgcggcgcca atgcattggg cccggtaccc agcttttgtt ccctttagtg agggttaatt
     1261 gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     1321 attccacaca acatacgagc cgggagcata aagtgtaaag cctggggtgc ctaatgagtg
     1381 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     1441 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     1501 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     1561 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     1621 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     1681 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     1741 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     1801 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     1861 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     1921 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     1981 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     2041 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     2101 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     2161 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     2221 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     2281 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     2341 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     2401 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     2461 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     2521 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     2581 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     2641 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     2701 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     2761 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     2821 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     2881 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     2941 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     3001 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     3061 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     3121 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     3181 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     3241 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     3301 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     3361 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     3421 aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     3481 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     3541 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     3601 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     3661 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     3721 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     3781 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     3841 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat
     3901 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagt
     3961 cgaccgccaa ttcaatatgg cgtatatgga ctcatgccaa ttcaatatgg tggatctgga
     4021 cctgtgccaa ttcaatatgg cgtatatgga ctcgtgccaa ttcaatatgg tggatctgga
     4081 ccccagccaa ttcaatatgg cggacttggc accatgccaa ttcaatatgg cggacctggc
     4141 actgtgccaa ctggggaggg gtctacttgg cacggtgcca agtttgagga ggggtcttgg
     4201 ccctgtgcca agtccgccat attgaattgg catggtgcca ataatggcgg ccatattggc
     4261 tatatgccag gatcaatata taggcaatat ccaatatggc cctatgccaa tatggctatt
     4321 ggccaggttc aatactatgt attggcccta tgccatatag tattccatat atgggttttc
     4381 ctattgacgt agatagcccc tcccaatggg cggtcccata taccatatat ggggcttcct
     4441 aataccgccc atagccactc ccccattgac gtcaatggtc tctatatatg gtctttccta
     4501 ttgacgtcat atgggcggtc ctattgacgt atatggcgcc tcccccattg acgtcaatta
     4561 cggtaaatgg cccgcctggc tcaatgccca ttgacgtcaa taggaccacc caccattgac
     4621 gtcaatggga tggctcattg cccattcata tccgttctca cgccccctat tgacgtcaat
     4681 gacggtaaat ggcccacttg gcagtacatc aatatctatt aatagtaact tggcaagtac
     4741 attactattg gaagtacgcc agggtacatt ggcagtactc ccattgacgt caatggcggt
     4801 aaatggcccg cgatggctgc caagtacatc cccattgacg tcaatgggga ggggcaatga
     4861 cgcaaatggg cgttccattg acgtaaatgg gcggtaggcg tgcctaatgg gaggtctata
     4921 taagcaatgc tcgtttaggg aac
//
