LOCUS       sequence-364246-        7286 bp    DNA     circular SYN 14-APR-2025
DEFINITION  GFP-tagged PKA RI-alpha subunit for mammalian expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7286)
  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 7286)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-EGFP
FEATURES             Location/Qualifiers
     source          1..7286
                     /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      2053..2062
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2059..2061,2062..2064,2065..2778)
                     /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(2104..2125)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2365..2384)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2712..2733
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2839..2857)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2840..2857)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2877..2894)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2883..3107
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3153..3581
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3240..3259)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3450..3471
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3590..3610)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3595..3924
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3775..3910
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3837..3856
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3991..4785
                     /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(4045..4064)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4655..4674
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4959..5080
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4996..5015)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5050..5069
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5129..5145)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5129..5145)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5142..5164)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5153..5169
                     /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(5177..5183,5184..5201,5202..5207))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5222..5243
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5360..5377)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5531..6119)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5611..5630)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6290..7081,7082..7150))
                     /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     6913..6932
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7151..7255)
                     /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 caccatggag
      901 tctggcagta ccgccgccag tgaggaggca cgcagccttc gagaatgtga gctctacgtc
      961 cagaagcata acattcaagc actgctcaaa gattctattg tgcagttgtg cactgctcga
     1021 cctgagagac ccatggcatt cctcagggaa tactttgaga ggttggagaa ggaggaggca
     1081 aaacagattc agaatctgca gaaagcaggc actcgtacag actcaaggga ggatgagatt
     1141 tctcctcctc cacccaaccc agtggttaaa ggtaggaggc gacgaggtgc tatcagcgct
     1201 gaggtctaca cggaggaaga tgcggcatcc tatgttagaa aggttatacc aaaagattac
     1261 aagacaatgg ccgctttagc caaagccatt gaaaagaatg tgctgttttc acatcttgat
     1321 gataatgaga gaagtgatat ttttgatgcc atgttttcgg tctcctttat cgcaggagag
     1381 actgtgattc agcaaggtga tgaaggggat aacttctatg tgattgatca aggagagacg
     1441 gatgtctatg ttaacaatga atgggcaacc agtgttgggg aaggagggag ctttggagaa
     1501 cttgctttga tttatggaac accgagagca gccactgtca aagcaaagac aaatgtgaaa
     1561 ttgtggggca tcgaccgaga cagctataga agaatcctca tgggaagcac actgagaaag
     1621 cggaagatgt atgaggaatt ccttagtaaa gtctctattt tagagtctct ggacaagtgg
     1681 gaacgtctta cggtagctga tgcattggaa ccagtgcagt ttgaagatgg gcagaagatt
     1741 gtggtgcagg gagaaccagg ggatgagttc ttcattattt tagaggggtc agctgctgtg
     1801 ctacaacgtc ggtcagaaaa tgaagagttt gttgaagtgg gaagattggg gccttctgat
     1861 tattttggtg aaattgcact actgatgaat cgtcctcgtg ctgccacagt tgttgctcgt
     1921 ggccccttga agtgcgttaa gctggaccga cctagatttg aacgtgttct tggcccatgc
     1981 tcagacatcc tcaaacgaaa catccagcag tacaacagtt ttgtgtcact gtctgtcgga
     2041 tccccaccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     2101 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     2161 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     2221 cccgtgccct ggcccaccct cgtgaccacc ctgagctacg gcgtgcagtg cttcagccgc
     2281 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     2341 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     2401 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     2461 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     2521 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     2581 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     2641 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
     2701 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     2761 gacgagctgt acaagtaaga attctgcaga tatccagcac agtggcggcc gctcgagcat
     2821 gcatctagag ggccctattc tatagtgtca cctaaatgct agagctcgct gatcagcctc
     2881 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     2941 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     3001 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     3061 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     3121 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     3181 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     3241 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     3301 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     3361 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     3421 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     3481 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     3541 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     3601 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3661 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3721 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     3781 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     3841 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     3901 tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct
     3961 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     4021 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     4081 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     4141 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     4201 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     4261 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     4321 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     4381 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     4441 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     4501 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     4561 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     4621 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     4681 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     4741 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     4801 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     4861 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     4921 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     4981 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     5041 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     5101 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     5161 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     5221 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5281 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5341 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5401 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5461 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5521 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5581 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5641 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5701 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5761 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5821 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5881 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5941 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     6001 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6061 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6121 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6181 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6241 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6301 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6361 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6421 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6481 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6541 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6601 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6661 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6721 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6781 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     6841 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6901 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6961 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     7021 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     7081 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     7141 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     7201 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     7261 ttccccgaaa agtgccacct gacgtc
//
