LOCUS       sequence-220174-        7173 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Mammalian expression of the mouse PKA catalytic subunit alpha 
            C-terminally tagged by monomeric mEGFP. Silent mutations were 
            introduced to make it resistant to shRNA knock-down..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7173)
  AUTHORS   Tillo SE, Xiong WH, Takahashi M, Miao S, Andrade AL, Fortin DA, Yang
            G, Qin M, Smoody BF, Stork PJS, Zhong H
  TITLE     Liberated PKA Catalytic Subunits Associate with the Membrane via 
            Myristoylation to Preferentially Phosphorylate Membrane Substrates.
  JOURNAL   Cell Rep. 2017 Apr 18;19(3):617-629. doi: 
            10.1016/j.celrep.2017.03.070.
  PUBMED    28423323
REFERENCE   2  (bases 1 to 7173)
  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.celrep.2017.03"; journalName: "Cell Rep"; date: 
            "2017-04-18- 18"; volume: "19"; issue: "3"; pages: "617-629"
COMMENT     Sequence Label: pCDNA3_shRNA resistant PKA-C-alpha-mEGFP
FEATURES             Location/Qualifiers
     source          1..7173
                     /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"
     CDS             1966..1992
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             join(1993..1995,1996..2709)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2035..2056)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2296..2315)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2643..2664
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2725..2743)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2726..2743)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2763..2780)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2769..2993
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3039..3467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3126..3145)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3336..3357
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3476..3496)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3481..3811
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3662..3797
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3724..3743
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3878..4672
                     /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(3932..3951)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4542..4561
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4846..4967
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4883..4902)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4937..4956
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5016..5032)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5016..5032)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5029..5051)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5040..5056
                     /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(5064..5070,5071..5088,5089..5094))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5109..5130
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5247..5264)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5418..6006)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5498..5517)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6177..6968,6969..7037))
                     /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     6800..6819
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7038..7142)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct 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 atgggtggac 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 ggagacccaa gcttgcggcc
      901 gctaccatgg gcaacgccgc cgccgccaag aagggcagcg agcaggagag cgtgaaagag
      961 ttcctagcca aagccaagga agatttcctg aaaaaatggg agaccccttc tcagaataca
     1021 gcccagttgg atcagtttga tagaatcaag acccttggca ccggctcctt tgggcgagtg
     1081 atgctggtga agcacaagga gagtgggaac cactacgcca tgaagatctt agacaagcag
     1141 aaggtggtga agctaaagca gatcgagcac actctgaatg agaagcgcat cctgcaggcc
     1201 gtcaacttcc cgttcctggt caagctggag ttttctttta aggacaactc aaacctgtac
     1261 atggtcatgg agtatgtagc tggtggcgag atgttctccc acctacggcg gattggaagg
     1321 ttcagcgagc cccatgcccg tttctacgcg gcgcagatcg tcctgacctt tgagtatctg
     1381 cactccctgg acctcatcta ccgggacctg aagcccgaga atcttctcat cgaccagcag
     1441 ggctatattc aggtgacaga cttcggtttt gccaagcgtg tgaaaggccg tacttggacc
     1501 ttgtgtggga cccctgagta cttggccccc gagattatcc tgagcaaagg ctacaacaag
     1561 gctgtggact ggtgggctct cggagtcctc atctacgaga tggctgctgg ttacccaccc
     1621 ttcttcgctg accagcctat ccagatctat gagaaaatcg tctctgggaa ggtgcggttc
     1681 ccatcccact tcagctctga cttgaaggac ctgctgcgga accttctgca agtggatctc
     1741 accaagcgct ttgggaacct caagaacggg gtcaatgaca tcaagaacca caagtggttt
     1801 gccacgactg actggattgc catctatcag agaaaggtgg aagctccctt cataccaaag
     1861 tttaaaggcc ctggggacac gagtaacttt gacgactatg aggaggaaga gatccgggtc
     1921 tccatcaatg agaagtgtgg caaggagttt actgagttta tcgattatga tgttcctgat
     1981 tatgcaagct taatgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     2041 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     2101 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     2161 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
     2221 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     2281 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     2341 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     2401 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     2461 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     2521 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     2581 gacaaccact acctgagcac ccagtccaag ctgagcaaag accccaacga gaagcgcgat
     2641 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     2701 tacaagtaat ctagagggcc ctattctata gtgtcaccta aatgctagag ctcgctgatc
     2761 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     2821 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     2881 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     2941 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     3001 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     3061 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     3121 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     3181 agctctaaat cggggcatcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     3241 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     3301 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     3361 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgg ggatttcggc
     3421 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     3481 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag gcaggcagaa gtatgcaaag
     3541 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     3601 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     3661 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     3721 ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg
     3781 aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt
     3841 cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     3901 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     3961 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     4021 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc
     4081 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     4141 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     4201 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     4261 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     4321 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     4381 cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca
     4441 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     4501 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     4561 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     4621 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
     4681 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc
     4741 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     4801 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     4861 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     4921 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     4981 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     5041 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     5101 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     5161 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     5221 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     5281 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     5341 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     5401 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     5461 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     5521 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5581 tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg
     5641 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5701 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5761 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5821 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     5881 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5941 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     6001 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     6061 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     6121 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     6181 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     6241 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     6301 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     6361 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     6421 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     6481 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6541 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6601 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6661 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6721 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6781 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6841 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     6901 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6961 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     7021 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     7081 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     7141 cgcacatttc cccgaaaagt gccacctgac gtc
//
