LOCUS       sequence-81965-e        4669 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shRNA against alpha-actinin-3 with eGFP transfection marker .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4669)
  AUTHORS   Schnizler MK, Schnizler K, Zha XM, Hall DD, Wemmie JA, Hell JW, 
            Welsh MJ
  TITLE     The cytoskeletal protein alpha-actinin regulates acid-sensing ion 
            channel 1a through a C-terminal interaction.
  JOURNAL   J Biol Chem. 2009 Jan 30;284(5):2697-705. doi: 
            10.1074/jbc.M805110200. Epub 2008 Nov 21.
  PUBMED    19028690
REFERENCE   2  (bases 1 to 4669)
  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: 
            "10.1074/jbc.M805110200"; journalName: "J Biol Chem"; date: 
            "2009-01-30- 30"; volume: "284"; issue: "5"; pages: "2697-705"
COMMENT     Sequence Label: pSIL-eGFP-shACTN3
FEATURES             Location/Qualifiers
     source          1..4669
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        84..387
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        388..591
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     542..562
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      630..639
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(636..638,639..641,642..1358)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYKY"
     primer_bind     complement(681..702)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(942..961)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1289..1310
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1367..1383
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1380..1693
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     1612..1632
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     complement(1771..1787)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1824..1844)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1824..1842)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1863..1879)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1863..1879)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1876..1898)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1887..1903
                     /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(1911..1917,1918..1935,1936..1941))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1956..1977
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2094..2111)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2265..2853)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2345..2364)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3024..3815,3816..3884))
                     /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     3647..3666
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3885..3989)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4015..4470)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4152..4173)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4364..4383
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4597..4619
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4611..4628
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4612..4628
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4638..4657
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4638..4656
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ctaaccgtat taccgccatg cattagttat taatagtaat caattacggg gtcattagtt
       61 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
      121 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
      181 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
      241 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      301 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      361 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt
      421 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
      481 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg
      541 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tggtttagtg
      601 aaccgtcaga tccgctagcg ctaccggtcg ccaccatggt gagcaagggc gaggagctgt
      661 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
      721 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
      781 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg
      841 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
      901 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
      961 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     1021 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
     1081 acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc
     1141 gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
     1201 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga
     1261 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     1321 ggatcactct cggcatggac gagctgtaca agtactaggt acccgctcta gaactagtgg
     1381 atccgacgcc gccatctcta ggcccgcgcc ggccccctcg cacagacttg tgggagaagc
     1441 tcggctactc ccctgccccg gttaatttgc atataatatt tcctagtaac tatagaggct
     1501 taatgtgcga taaaagacag ataatctgtt ctttttaata ctagctacat tttacatgat
     1561 aggcttggat ttctataaga gatacaaata ctaaattatt attttaaaaa acagcacaaa
     1621 aggaaactca ccctaactgt aaagtaattg tgtgttttga gactataaat atcccttgga
     1681 gaaaagcctt gtttgggcca acgagaagct gatggaggtt caagagacct ccatcagctt
     1741 ctcgtttttt ttaattcctg cagcccgggg gatccactag ttctagagcg gccgccaccg
     1801 cggtggagct ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca
     1861 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     1921 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     1981 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     2041 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     2101 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     2161 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     2221 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     2281 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     2341 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     2401 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     2461 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     2521 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     2581 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     2641 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     2701 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     2761 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     2821 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     2881 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     2941 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     3001 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     3061 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     3121 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     3181 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     3241 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     3301 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     3361 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     3421 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     3481 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     3541 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     3601 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     3661 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     3721 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     3781 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     3841 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     3901 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     3961 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg
     4021 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     4081 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     4141 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     4201 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     4261 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     4321 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     4381 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     4441 ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg cgcaactgtt
     4501 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
     4561 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
     4621 cggccagtga gcgcgcgtaa tacgactcac tatagggcga attgggtac
//
