LOCUS       sequence-127238-        4995 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GFP reporter protein fused to three GSK3 phosphorylation sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4995)
  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 4995)
  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-GSK3-MAPK
FEATURES             Location/Qualifiers
     source          1..4995
                     /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(1564..1581)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1735..2323)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1815..1834)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2494..3285,3286..3354))
                     /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     3117..3136
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3355..3459)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3485..3940)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3622..3643)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3834..3853
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4011..4995
                     /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 cttctgctgc cgcttctgct gccgcttctg 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 tcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     1561 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     1621 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     1681 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     1741 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     1801 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     1861 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     1921 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     1981 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     2041 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     2101 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     2161 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     2221 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     2281 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     2341 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     2401 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     2461 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     2521 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     2581 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     2641 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     2701 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     2761 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     2821 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     2881 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     2941 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     3001 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     3061 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     3121 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     3181 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     3241 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     3301 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     3361 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     3421 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     3481 acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     3541 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     3601 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     3661 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     3721 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     3781 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     3841 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     3901 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg
     3961 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gtcgaccgcc
     4021 aattcaatat ggcgtatatg gactcatgcc aattcaatat ggtggatctg gacctgtgcc
     4081 aattcaatat ggcgtatatg gactcgtgcc aattcaatat ggtggatctg gaccccagcc
     4141 aattcaatat ggcggacttg gcaccatgcc aattcaatat ggcggacctg gcactgtgcc
     4201 aactggggag gggtctactt ggcacggtgc caagtttgag gaggggtctt ggccctgtgc
     4261 caagtccgcc atattgaatt ggcatggtgc caataatggc ggccatattg gctatatgcc
     4321 aggatcaata tataggcaat atccaatatg gccctatgcc aatatggcta ttggccaggt
     4381 tcaatactat gtattggccc tatgccatat agtattccat atatgggttt tcctattgac
     4441 gtagatagcc cctcccaatg ggcggtccca tataccatat atggggcttc ctaataccgc
     4501 ccatagccac tcccccattg acgtcaatgg tctctatata tggtctttcc tattgacgtc
     4561 atatgggcgg tcctattgac gtatatggcg cctcccccat tgacgtcaat tacggtaaat
     4621 ggcccgcctg gctcaatgcc cattgacgtc aataggacca cccaccattg acgtcaatgg
     4681 gatggctcat tgcccattca tatccgttct cacgccccct attgacgtca atgacggtaa
     4741 atggcccact tggcagtaca tcaatatcta ttaatagtaa cttggcaagt acattactat
     4801 tggaagtacg ccagggtaca ttggcagtac tcccattgac gtcaatggcg gtaaatggcc
     4861 cgcgatggct gccaagtaca tccccattga cgtcaatggg gaggggcaat gacgcaaatg
     4921 ggcgttccat tgacgtaaat gggcggtagg cgtgcctaat gggaggtcta tataagcaat
     4981 gctcgtttag ggaac
//
