LOCUS       sequence-364318-        6449 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EGFP-tagged docking/dimerization (D/D) and linker region of PKA 
            RI-alpha; for mammalian expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6449)
  AUTHORS   Zhang JZ, Lu TW, Stolerman LM, Tenner B, Yang JR, Zhang JF, Falcke 
            M, Rangamani P, Taylor SS, Mehta S, Zhang J
  TITLE     Phase Separation of a PKA Regulatory Subunit Controls cAMP 
            Compartmentation and Oncogenic Signaling.
  JOURNAL   Cell. 2020 Sep 17;182(6):1531-1544.e15. doi: 
            10.1016/j.cell.2020.07.043. Epub 2020 Aug 25.
  PUBMED    32846158
REFERENCE   2  (bases 1 to 6449)
  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.cell.2020.07.043"; journalName: "Cell"; date: 
            "2020-09-17- 17"; volume: "182"; issue: "6"; pages: "1531-1544.e15"
COMMENT     Sequence Label: pcDNA3.1(+)-RI-alpha(D/D+linker)-EGFP
FEATURES             Location/Qualifiers
     source          1..6449
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      889..898
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1216..1225
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1222..1224,1225..1227,1228..1941)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1267..1288)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1528..1547)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1875..1896
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2002..2020)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2003..2020)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2040..2057)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2046..2270
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2316..2744
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2403..2422)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2613..2634
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2753..2773)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2758..3087
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2938..3073
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3000..3019
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3154..3948
                     /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(3208..3227)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3818..3837
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4122..4243
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4159..4178)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4213..4232
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4292..4308)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4292..4308)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4305..4327)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4316..4332
                     /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(4340..4346,4347..4364,4365..4370))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4385..4406
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4523..4540)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4694..5282)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4774..4793)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5453..6244,6245..6313))
                     /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     6076..6095
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6314..6418)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccgc caccatggca
      901 cgcagccttc gagaatgtga gctctacgtc cagaagcata acattcaagc actgctcaaa
      961 gattctattg tgcagttgtg cactgctcga cctgagagac ccatggcatt cctcagggaa
     1021 tactttgaga ggttggagaa ggaggaggca aaacagattc agaatctgca gaaagcaggc
     1081 actcgtacag actcaaggga ggatgagatt tctcctcctc cacccaaccc agtggttaaa
     1141 ggtaggaggc gacgaggtgc tatcagcgct gaggtctaca cggaggaaga tgcggcatcc
     1201 ggatccccac cggtcgccac catggtgagc aagggcgagg agctgttcac cggggtggtg
     1261 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
     1321 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     1381 ctgcccgtgc cctggcccac cctcgtgacc accctgagct acggcgtgca gtgcttcagc
     1441 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     1501 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     1561 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     1621 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
     1681 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
     1741 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     1801 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
     1861 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     1921 atggacgagc tgtacaagta agaattctgc agatatccag cacagtggcg gccgctcgag
     1981 catgcatcta gagggcccta ttctatagtg tcacctaaat gctagagctc gctgatcagc
     2041 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     2101 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     2161 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     2221 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     2281 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     2341 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     2401 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2461 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2521 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2581 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2641 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     2701 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     2761 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     2821 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     2881 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     2941 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     3001 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     3061 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     3121 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     3181 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     3241 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     3301 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
     3361 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     3421 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     3481 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     3541 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     3601 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     3661 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     3721 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     3781 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     3841 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     3901 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     3961 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     4021 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     4081 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     4141 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     4201 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     4261 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     4321 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     4381 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4441 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4501 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4561 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4621 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4681 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4741 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4801 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4861 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4921 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4981 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     5041 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5101 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     5161 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5221 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5281 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5341 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5401 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5461 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5521 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5581 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5641 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5701 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5761 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5821 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     5881 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     5941 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     6001 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     6061 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6121 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6181 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6241 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6301 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6361 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6421 catttccccg aaaagtgcca cctgacgtc
//
