LOCUS       sequence-237874-        6405 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Homology arms and linker-mEGFP sequence for C-terminus tagging of 
            human ACTN2, via excisable Cas9-excisable CAGGS-mCherry selection 
            cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 6405)
  AUTHORS   Allen Institute for Cell Science
  TITLE     Allen Institute for Cell Science Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6405)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: AICSDP-63: ACTN2-mEGFP
FEATURES             Location/Qualifiers
     source          1..6405
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     polyA_signal    1091..1212
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1128..1147)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1182..1201
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     enhancer        1228..1607
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1609..1886
                     /label=chicken beta-actin promoter
     intron          1888..2905
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2829..2851
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2913..2932
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(3139..3157)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3407..3426
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3717..3770
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3771..4370
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3771..3790)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4267..4287
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4387..4611
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             join(4692..4694,4695..5408)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4734..4755)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4995..5014)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5342..5363
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 tagccgttca gtcagtaaaa aattggcaag cttttcatta catgtagcct agaccttcag
       61 gaggagttta attgccagtt cagaattgta atattttacc ccccaaacaa taaaaacatc
      121 tctatgtata aattcaattt gggacaggat tattttaccc tgttttctgg ctgagaggat
      181 aatgaacatt accacaagga ggttagcatg ctaaaacaca gtaacagagc catatttatt
      241 accttaaaaa tcagataaga ccaactattt ttaaaataat gttttcttta aaatgaatca
      301 tttacccatg gggtgagggt ggatgggaaa tagcccagct gatttagaag gtaagaaacc
      361 atggctcctc caaccccact aagaaacttc tactataaat tatataatat gcaaactata
      421 atgatataaa ttataatgtg atatttgaga tttacttatt ttgactttta ccaaccagac
      481 tatttggctg gaattgtcct atttcccact gaactttttt tttaaaagct tcatcttttc
      541 tggtatgaaa tgcagatcat agtacgtatc ctcgcattgt ggctcagttt gagaactact
      601 agtaatgctc catttgcctt tatgaagcat atcacccttc ctgctggcca ggccgcttct
      661 agatacgctc ctacaagtaa aacttggctt tctgttggtt gtctggtact actatgccaa
      721 tagactccct attctttagt ccttttaaaa aattaaacag atgcaagaaa tatgtaagta
      781 ttaagactgt ttatgttgtg gtgtttctgc aactgactgc aaacacgtgt gtattttttc
      841 ccagccatac atcctggcgg aggagctgcg tcgggagctg cccccggatc aggcccagta
      901 ctgcatcaag aggatgcccg cctactcggg cccaggcagt gtgcctggtg cactggatta
      961 cgctgcgttc tcttccgcac tgtacggcga gagcgatctg gtcgacggga ctgctgggcc
     1021 cggagaggtt cccgacatac caataacttc gtatagtgta ggatatacga agttatatta
     1081 gtattccgat aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     1141 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     1201 caatgtatct tatcatgtct gagtctcgac attgattatt gactagttat taatagtaat
     1261 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
     1321 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
     1381 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     1441 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     1501 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     1561 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtgagcc
     1621 ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat
     1681 ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg cgcgcgccag
     1741 gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca
     1801 atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct
     1861 ataaaaagcg aagcgcgcgg cgggcgggga gtcgctgcga cgctgccttc gccccgtgcc
     1921 ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca
     1981 ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagcgcttgg tttaatgacg
     2041 gcttgtttct tttctgtggc tgcgtgaaag ccttgagggg ctccgggagg gccctttgtg
     2101 cggggggagc ggctcggggg gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg
     2161 ctccgcgctg cccggcggct gtgagcgctg cgggcgcggc gcggggcttt gtgcgctccg
     2221 cagtgtgcgc gaggggagcg cggccggggg cggtgccccg cggtgcgggg ggggctgcga
     2281 ggggaacaaa ggctgcgtgc ggggtgtgtg cgtggggggg tgagcagggg gtgtgggcgc
     2341 gtcggtcggg ctgcaacccc ccctgcaccc ccctccccga gttgctgagc acggcccggc
     2401 ttcgggtgcg gggctccgta cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg
     2461 gcggcaggtg ggggtgccgg gcggggcggg gccgcctcgg gccggggagg gctcggggga
     2521 ggggcgcggc ggcccccgga gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg
     2581 ccttttatgg taatcgtgcg agagggcgca gggacttcct ttgtcccaaa tctgtgcgga
     2641 gccgaaatct gggaggcgcc gccgcacccc ctctagcggg cgcggggcga agcggtgcgg
     2701 cgccggcagg aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct
     2761 tctccctctc cagcctcggg gctgtccgcg gggggacggc tgccttcggg ggggacgggg
     2821 cagggcgggg ttcggcttct ggcgtgtgac cggcggctct agagcctctg ctaaccatgt
     2881 tcatgccttc ttctttttcc tacagctcct gggcaacgtg ctggttattg tgctgtctca
     2941 tcattttggc aaagaattcc gccaccgggg atccaccgtt cgaagagctt accatggtct
     3001 ccaaaggaga agaagataac atggccatca tcaaggagtt catgcgcttc aaggtgcaca
     3061 tggagggctc cgtgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct
     3121 acgagggcac ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttcgcct
     3181 gggacatcct gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag caccccgccg
     3241 acatccccga ctacttgaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga
     3301 acttcgagga cggcggcgtg gtgaccgtga cccaggactc ctccctgcag gacggcgagt
     3361 tcatctacaa ggtgaagctg cgcggcacca acttcccctc cgacggcccc gtaatgcaga
     3421 agaagaccat gggctgggag gcctcctccg agcggatgta ccccgaggac ggcgccctga
     3481 agggcgagat caagcagagg ctgaagctga aggacggcgg ccactacgac gctgaggtca
     3541 agaccaccta caaggccaag aagcccgtgc agctgcccgg cgcctacaac gtcaacatca
     3601 agttggacat cacctcccac aacgaggact acaccatcgt ggaacagtac gaacgcgccg
     3661 agggccgcca ctccaccggc ggaatggatg aactctataa agcggccgca cttaaggagg
     3721 gcagaggaag tctgctcaca tgcggtgacg tcgaggagaa tcctggccca atgaccgagt
     3781 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg
     3841 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg
     3901 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg
     3961 tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag
     4021 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg
     4081 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt
     4141 tcctggccac cgtcggagtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg
     4201 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg
     4261 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg
     4321 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga cttaagtgag
     4381 gtactcctgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     4441 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     4501 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     4561 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gaaataactt
     4621 cgtatagtgt aggatatacg aagttatata ggtatgtcgg gaacctctcc gggcccggat
     4681 ccattgctac tgtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     4741 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     4801 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     4861 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     4921 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     4981 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     5041 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     5101 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     5161 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     5221 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     5281 acaaccacta cctgagcacc cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc
     5341 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     5401 acaagtgatg ctgagcttct gtaatcactc atggcatcag aatgcaataa aagcggaagt
     5461 cacagtttgt ttcctggaaa ctttgacaag ctttattaag ttgagagaga gagaggggaa
     5521 aaaaaaaagc ctttcgtagt tcagtaattg ccagcaatat aacacggcta aaatgaagtt
     5581 tttacagtat atgacatagt gcgcttcata aataggttta tttctgagtt tttagcaaaa
     5641 tgtaatgaaa tatcaggttg atttctttga ttaaacagaa caaattactt gagtaatagg
     5701 aaattaggag gatctaggga cagaaggaaa gtgaaaaatg tgaaaataca aaatacccaa
     5761 gatttaagac cggggggaaa aaaccacaaa ttggtaaata aaggtttgct atttgtaaaa
     5821 aatttcattt atctctaata tgcttatgtg attgccccta ggggagtata tttgggattc
     5881 taatgtttta ttttcatgct tatccaaaga ttactattgt atcttcaaat gaatttaata
     5941 ttgtgagatg gaactgccgg ggattaaaaa gactacccaa aagatttttg gcacttacaa
     6001 tttttaaaat agtttatgtc atctcttcat tatttagggc tggatggtca actcagtcag
     6061 tgattttttg atgcttctct tatcctccag aatagagacc taaggacacg tggaagtcag
     6121 tttaattgcc agagagaagg atgcaatcac taggtaaaat gaggttttta ggattattta
     6181 ttgattccag gttcccatgc tttttgttag agcttattag tacaggttct caagagatga
     6241 ccacataaaa gtgctctgtt tataaataag caggtttctg tagtactgac tggttcatca
     6301 caaggcaagt cagaaaccag tatccttcta gctctccagt caggacttcc ttatgcctct
     6361 agttttatga ccggttaagg agaagccaga gttagagtag gagag
//
