LOCUS       sequence-224093-        5764 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of shRNA for knocking down endogenous PKA-C-alpha. EGFP 
            is also expressed..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5764)
  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 5764)
  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.celrep.2017.03"; journalName: "Cell Rep"; date: 
            "2017-04-18- 18"; volume: "19"; issue: "3"; pages: "617-629"
COMMENT     Sequence Label: pEFU6-EGFP_shPKA-C-alpha
FEATURES             Location/Qualifiers
     source          1..5764
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        52..1230
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          283..1221
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1178..1198
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     misc_feature    1232..1312
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      1314..1323
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1320..1322,1323..1325,1326..2039)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1320..1322,1323..1325,1326..2036)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1365..1386)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1626..1645)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1973..1994
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2162..2283
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2199..2218)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2253..2272
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2290..2745)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2427..2448)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2639..2658
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2772..2876
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2878..3235
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2878..3069
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2901..2921)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3086..3221
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3270..4064
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3270..4064
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3324..3343)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3934..3953
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4252..4271)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4296..4343
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4672..5260
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5161..5180
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        complement(5459..5707)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(5517..5536)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(5687..5707)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 ttttgcctgc gcgtctttcc actgggggaa ttcactacgc gttaatcgtg aggctccggt
       61 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc
      121 ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
      181 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
      241 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt
      301 tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc
      361 tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg
      421 aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc
      481 tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag
      541 tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt
      601 cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc
      661 gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca
      721 ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg
      781 ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga
      841 gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc
      901 tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc
      961 gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg
     1021 agcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc
     1081 cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg
     1141 gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa
     1201 agtttttttc ttccatttca ggtgtcgtga ggctagcgct accggactca gatctcgagc
     1261 tcaagctacg aattctgcag tcgacggtac cgcgggcccg cgatccaccg gtcgccacca
     1321 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     1381 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     1441 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     1501 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     1561 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     1621 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     1681 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     1741 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     1801 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     1861 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     1921 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     1981 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaaa
     2041 gcggccgcga ctctagatca taatcagcca taccacattt gtagaggttt tacttgcttt
     2101 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     2161 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     2221 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     2281 ttaaggcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     2341 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     2401 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     2461 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     2521 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     2581 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     2641 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     2701 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc
     2761 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     2821 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtcctg
     2881 aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     2941 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     3001 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3061 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     3121 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     3181 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg
     3241 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     3301 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     3361 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     3421 ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg
     3481 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     3541 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     3601 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     3661 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     3721 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     3781 tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     3841 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     3901 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     3961 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     4021 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     4081 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     4141 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     4201 gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg
     4261 gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac
     4321 gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt
     4381 cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc
     4441 ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc
     4501 tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt
     4561 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     4621 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     4681 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     4741 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     4801 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     4861 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     4921 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     4981 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     5041 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     5101 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     5161 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     5221 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     5281 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     5341 tgattctgtg gataaccgta ttaccgccat gcattagtta ttaactagtg ccaagctttt
     5401 ccaaaaaaaa cttgaattct ccttcaatct cttgaattga aggagaattc aagttcggga
     5461 tcccgcgtcc tttccacaag atatataaac ccaagaaatc gaaatacttt caagttacgg
     5521 taagcatatg atagtccatt ttaaaacata attttaaaac tgcaaactac ccaagaaatt
     5581 attactttct acgtcacgta ttttgtacta atatctttgt gtttacagtc aaattaattc
     5641 taattatctc tctaacagcc ttgtatcgta tatgcaaata tgaaggaatc atgggaaata
     5701 ggccctcttc ctgcccgacc ttggcgcgcg ctcggcgcgc ggtcacgctc cgtcacgtgg
     5761 tgcg
//
