LOCUS       sequence-332539-        6185 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Mammalian expression of the N-terminal 15 residues of mouse PKA 
            catalytic subunit alpha C-terminally tagged by monomeric Venus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6185)
  AUTHORS   Xiong WH, Qin M, Zhong H
  TITLE     Myristoylation alone is sufficient for PKA catalytic subunits to 
            associate with the plasma membrane to regulate neuronal functions.
  JOURNAL   Proc Natl Acad Sci U S A. 2021 Apr 13;118(15). pii: 2021658118. doi:
            10.1073/pnas.2021658118.
  PUBMED    33876760
REFERENCE   2  (bases 1 to 6185)
  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"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2021-04-13"; volume: "118(15). pii: 
            2021658118. doi"; pages: " 10.1073/pnas.2021658118"
COMMENT     Sequence Label: pCDNA3_PKA-C-alpha-N15-mVenus
FEATURES             Location/Qualifiers
     source          1..6185
                     /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             958..984
                     /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(985..987,988..1701)
                     /codon_start=1
                     /product="Venus YFP with monomerizing A206K mutation (Nagai
                     et al., 2002; Kremers et al., 2006)"
                     /label=mVenus
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     LICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1027..1048)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1288..1307)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1635..1656
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(1738..1756)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1739..1756)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1776..1793)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1782..2006
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2052..2480
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2139..2158)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2349..2370
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2489..2509)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2494..2823
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2674..2809
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2736..2755
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2890..3684
                     /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(2944..2963)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3554..3573
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3858..3979
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3895..3914)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3949..3968
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4028..4044)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4028..4044)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4041..4063)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4052..4068
                     /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(4076..4082,4083..4100,4101..4106))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4121..4142
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4259..4276)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4430..5018)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4510..4529)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5189..5980,5981..6049))
                     /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     5812..5831
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6050..6154)
                     /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 ggagacccaa gcttgcggcc
      901 gctaccatgg gcaacgccgc cgccgccaag aagggcagcg agcaggagag catcgattat
      961 gatgttcctg attatgcaag cttaatgagc aagggcgagg agctgttcac cggggtggtg
     1021 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
     1081 ggcgagggcg atgccaccta cggcaagctg accctgaagc tgatctgcac caccggcaag
     1141 ctgcccgtgc cctggcccac cctcgtgacc accctgggct acggcctgca gtgcttcgcc
     1201 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     1261 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     1321 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     1381 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
     1441 accgccgaca agcagaagaa cggcatcaag gccaacttca agatccgcca caacatcgag
     1501 gacggcggcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     1561 gtgctgctgc ccgacaacca ctacctgagc taccagtcca agctgagcaa agaccccaac
     1621 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     1681 atggacgagc tgtacaagta agaattcaag gcctctcgag cctctagagg gccctattct
     1741 atagtgtcac ctaaatgcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca
     1801 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     1861 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     1921 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     1981 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     2041 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     2101 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     2161 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     2221 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     2281 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     2341 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     2401 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     2461 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     2521 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     2581 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     2641 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     2701 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     2761 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     2821 aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga
     2881 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     2941 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     3001 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     3061 aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     3121 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     3181 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     3241 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     3301 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     3361 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc
     3421 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     3481 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     3541 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     3601 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     3661 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     3721 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     3781 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     3841 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     3901 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     3961 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
     4021 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     4081 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     4141 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     4201 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     4261 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     4321 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     4381 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     4441 tccgcccccc tgacgagcat tacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     4501 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     4561 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     4621 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4681 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4741 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4801 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4861 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4921 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4981 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     5041 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     5101 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     5161 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     5221 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     5281 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     5341 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     5401 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     5461 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     5521 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     5581 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     5641 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     5701 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     5761 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     5821 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     5881 tctcaaggat cttaccgctg ttgagatcca gttcaatgta acccactcgt gcacccaact
     5941 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     6001 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     6061 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     6121 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     6181 acgtc
//
