LOCUS       sequence-215471-        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        complement(85..333)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(143..162)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(313..333)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     promoter        442..1620
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          673..1611
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1568..1588
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     misc_feature    1622..1702
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      1704..1713
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1710..1712,1713..1715,1716..2429)
                     /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(1710..1712,1713..1715,1716..2426)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1755..1776)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2016..2035)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2363..2384
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2552..2673
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2589..2608)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2643..2662
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2680..3135)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2817..2838)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3029..3048
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3162..3266
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3268..3625
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3268..3459
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3291..3311)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3476..3611
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3660..4454
                     /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             3660..4454
                     /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(3714..3733)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4324..4343
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4642..4661)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4686..4733
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5062..5650
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5551..5570
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa ctagtgccaa gcttttccaa aaaaaacttg aattctcctt caatctcttg
       61 aattgaagga gaattcaagt tcgggatccc gcgtcctttc cacaagatat ataaacccaa
      121 gaaatcgaaa tactttcaag ttacggtaag catatgatag tccattttaa aacataattt
      181 taaaactgca aactacccaa gaaattatta ctttctacgt cacgtatttt gtactaatat
      241 ctttgtgttt acagtcaaat taattctaat tatctctcta acagccttgt atcgtatatg
      301 caaatatgaa ggaatcatgg gaaataggcc ctcttcctgc ccgaccttgg cgcgcgctcg
      361 gcgcgcggtc acgctccgtc acgtggtgcg ttttgcctgc gcgtctttcc actgggggaa
      421 ttcactacgc gttaatcgtg aggctccggt gcccgtcagt gggcagagcg cacatcgccc
      481 acagtccccg agaagttggg gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg
      541 cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg
      601 ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc
      661 gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat
      721 ggcccttgcg tgccttgaat tacttccacc tggctgcagt acgtgattct tgatcccgag
      781 cttcgggttg gaagtgggtg ggagagttcg aggccttgcg cttaaggagc cccttcgcct
      841 cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc gcgtgcgaat ctggtggcac
      901 cttcgcgcct gtctcgctgc tttcgataag tctctagcca tttaaaattt ttgatgacct
      961 gctgcgacgc tttttttctg gcaagatagt cttgtaaatg cgggccaaga tctgcacact
     1021 ggtatttcgg tttttggggc cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt
     1081 tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg gacgggggta gtctcaagct
     1141 ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta tcgccccgcc ctgggcggca
     1201 aggctggccc ggtcggcacc agttgcgtga gcggaaagat ggccgcttcc cggccctgct
     1261 gcagggagct caaaatggag gacgcggcgc tcgggagagc gggcgggtga gtcacccaca
     1321 caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg
     1381 gcgccgtcca ggcacctcga ttagttctcg agcttttgga gtacgtcgtc tttaggttgg
     1441 ggggaggggt tttatgcgat ggagtttccc cacactgagt gggtggagac tgaagttagg
     1501 ccagcttggc acttgatgta attctccttg gaatttgccc tttttgagtt tggatcttgg
     1561 ttcattctca agcctcagac agtggttcaa agtttttttc ttccatttca ggtgtcgtga
     1621 ggctagcgct accggactca gatctcgagc tcaagctacg aattctgcag tcgacggtac
     1681 cgcgggcccg cgatccaccg gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg
     1741 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     1801 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     1861 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     1921 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     1981 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     2041 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     2101 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     2161 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     2221 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     2281 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     2341 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     2401 ctctcggcat ggacgagctg tacaagtaaa gcggccgcga ctctagatca taatcagcca
     2461 taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct
     2521 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta
     2581 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     2641 ttgtggtttg tccaaactca tcaatgtatc ttaaggcgta aattgtaagc gttaatattt
     2701 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     2761 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     2821 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     2881 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     2941 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     3001 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     3061 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     3121 cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     3181 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     3241 ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg
     3301 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     3361 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     3421 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     3481 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     3541 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     3601 ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga tcgtttcgca
     3661 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     3721 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     3781 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     3841 aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     3901 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     3961 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     4021 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     4081 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     4141 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg
     4201 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     4261 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     4321 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     4381 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     4441 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct
     4501 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     4561 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     4621 ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc
     4681 tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg ttcataaacg
     4741 cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca
     4801 atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg
     4861 gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact catatatact
     4921 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     4981 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     5041 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     5101 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     5161 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     5221 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     5281 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     5341 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     5401 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     5461 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     5521 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     5581 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     5641 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     5701 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat
     5761 gcat
//
