LOCUS       sequence-224274-        7172 bp    DNA     circular SYN 15-APR-2025
DEFINITION  The G2A mutant 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 7172)
  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 7172)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 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 nmPKA-C-alpha-mEGFP
FEATURES             Location/Qualifiers
     source          1..7172
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        171..550
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        551..754
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     705..725
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     799..818
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        799..817
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1902..1928
                     /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(1929..1931,1932..2645)
                     /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(1971..1992)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2232..2251)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2579..2600
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2661..2679)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2662..2679)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2699..2716)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2705..2929
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2975..3403
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3062..3081)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3272..3293
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3412..3432)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3417..3746
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3597..3732
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3659..3678
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3813..4607
                     /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(3867..3886)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4477..4496
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4781..4902
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4818..4837)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4872..4891
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4951..4967)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4951..4967)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4964..4986)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4975..4991
                     /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(4999..5005,5006..5023,5024..5029))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5044..5065
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5182..5199)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5353..5941)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(6112..6903,6904..6972))
                     /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     6735..6754
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6973..7077)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7152..7171)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag
       61 caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag
      121 ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt
      181 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
      241 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
      301 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
      361 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
      421 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
      481 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      541 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
      601 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
      661 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
      721 gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc
      781 ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt gcggccgcta
      841 ccatggccaa cgccgccgcc gccaagaagg gcagcgagca ggagagcgtg aaagagttcc
      901 tagccaaagc caaggaagat ttcctgaaaa aatgggagac cccttctcag aatacagccc
      961 agttggatca gtttgataga atcaagaccc ttggcaccgg ctcctttggg cgagtgatgc
     1021 tggtgaagca caaggagagt gggaaccact acgccatgaa gatcttagac aagcagaagg
     1081 tggtgaagct aaagcagatc gagcacactc tgaatgagaa gcgcatcctg caggccgtca
     1141 acttcccgtt cctggtcaag ctggagtttt cttttaagga caactcaaac ctgtacatgg
     1201 tcatggagta tgtagctggt ggcgagatgt tctcccacct acggcggatt ggaaggttca
     1261 gcgagcccca tgcccgtttc tacgcggcgc agatcgtcct gacctttgag tatctgcact
     1321 ccctggacct catctaccgg gacctgaagc ccgagaatct tctcatcgac cagcagggct
     1381 atattcaggt gacagacttc ggttttgcca agcgtgtgaa aggccgtact tggaccttgt
     1441 gtgggacccc tgagtacttg gcccccgaga ttatcctgag caaaggctac aacaaggctg
     1501 tggactggtg ggctctcgga gtcctcatct acgagatggc tgctggttac ccacccttct
     1561 tcgctgacca gcctatccag atctatgaga aaatcgtctc tgggaaggtg cggttcccat
     1621 cccacttcag ctctgacttg aaggacctgc tgcggaacct tctgcaagtg gatctcacca
     1681 agcgctttgg gaacctcaag aacggggtca atgacatcaa gaaccacaag tggtttgcca
     1741 cgactgactg gattgccatc tatcagagaa aggtggaagc tcccttcata ccaaagttta
     1801 aaggccctgg ggacacgagt aactttgacg actatgagga ggaagagatc cgggtctcca
     1861 tcaatgagaa gtgtggcaag gagtttactg agtttatcga ttatgatgtt cctgattatg
     1921 caagcttaat gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     1981 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     2041 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     2101 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     2161 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     2221 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     2281 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     2341 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     2401 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     2461 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     2521 accactacct gagcacccag tccaagctga gcaaagaccc caacgagaag cgcgatcaca
     2581 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     2641 agtaatctag agggccctat tctatagtgt cacctaaatg ctagagctcg ctgatcagcc
     2701 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     2761 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     2821 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     2881 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
     2941 gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
     3001 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     3061 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     3121 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     3181 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     3241 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     3301 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcccat
     3361 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt
     3421 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3481 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     3541 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     3601 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     3661 tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct
     3721 tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat
     3781 ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     3841 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     3901 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     3961 agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc
     4021 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     4081 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     4141 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     4201 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     4261 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     4321 tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg
     4381 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     4441 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     4501 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     4561 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg
     4621 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc
     4681 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     4741 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     4801 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     4861 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     4921 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     4981 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     5041 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5101 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5161 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     5221 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5281 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5341 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     5401 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc ctcctggaag
     5461 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     5521 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     5581 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     5641 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     5701 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     5761 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     5821 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     5881 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     5941 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     6001 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     6061 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     6121 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     6181 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     6241 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     6301 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     6361 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     6421 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     6481 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     6541 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     6601 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     6661 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     6721 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     6781 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     6841 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     6901 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     6961 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     7021 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     7081 atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg
     7141 tgcactctca gtacaatctg ctctgatgcc gc
//
