LOCUS       sequence-330059-        7439 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses an ACTB gene fragment with P2A, flanked by mCherry and 
            EGFP, in Mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7439)
  AUTHORS   Zou RS, Liu Y, Wu B, Ha T
  TITLE     Cas9 deactivation with photocleavable guide RNAs.
  JOURNAL   Mol Cell. 2021 Apr 1;81(7):1553-1565.e8. doi: 
            10.1016/j.molcel.2021.02.007. Epub 2021 Mar 3.
  PUBMED    33662274
REFERENCE   2  (bases 1 to 7439)
  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.molcel.2021.02.007"; journalName: "Mol Cell"; date: 
            "2021-04-1- 1"; volume: "81"; issue: "7"; pages: "1553-1565.e8"
COMMENT     Sequence Label: pLPC-mCh-ACTB-P2A-EGFP
FEATURES             Location/Qualifiers
     source          1..7439
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..470
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    535..734
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             935..1351
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1281..1303
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1319..1335
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     CDS             1369..1968
                     /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(1369..1388)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1865..1885
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(2057..2076)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2248..2627
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2628..2831
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2782..2802
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2876..2895
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2876..2894
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2928..2952
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      2973..2982
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2979..3686
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3124..3142)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3392..3411
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3849..3905
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(3906..3908,3909..3911,3912..4625)
                     /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(3906..3908,3909..3911,3912..4622)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3951..3972)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4212..4231)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4559..4580
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(4648..4672)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     LTR             4926..5395
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5525..5542)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5696..6284)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5776..5795)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6444..7235,7236..7304))
                     /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     7067..7086
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7305..7409)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa
       61 gaacagatgg aacagctgaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc
      121 ccggctcagg gccaagaaca gatggtcccc agatgcggtc cagccctcag cagtttctag
      181 agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg
      241 aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat
      301 aaaagagccc acaacccctc actcggggcg ccagtcctcc gattgactga gtcgcccggg
      361 tacccgtgta tccaataaac cctcttgcag ttgcatccga cttgtggtct cgctgttcct
      421 tgggagggtc tcctctgagt gattgactac ccgtcagcgg gggtctttca tttgggggct
      481 cgtccgggat cgggagaccc ctgcccaggg accaccgacc caccaccggg aggcaagctg
      541 gccagcaact tatctgtgtc tgtccgattg tctagtgtct atgactgatt ttatgcgcct
      601 gcgtcggtac tagttagcta actagctctg tatctggcgg acccgtggtg gaactgacga
      661 gttctgaaca cccggccgca accctgggag acgtcccagg gactttgggg gccgtttttg
      721 tggcccgacc tgaggaaggg agtcgatgtg gaatccgacc ccgtcaggat atgtggttct
      781 ggtaggagac gagaacctaa aacagttccc gcctccgtct gaatttttgc tttcggtttg
      841 gaaccgaagc cgcgcgtctt gtctgctgca gcgctgcagc atcgttctgt gttgtctctg
      901 tctgactgtg tttctgtatt tgtctgaaaa ttagggccag actgttacca ctcccttaag
      961 tttgacctta ggtcactgga aagatgtcga gcggatcgct cacaaccagt cggtagatgt
     1021 caagaagaga cgttgggtta ccttctgctc tgcagaatgg ccaaccttta acgtcggatg
     1081 gccgcgagac ggcaccttta accgagacct catcacccag gttaagatca aggtcttttc
     1141 acctggcccg catggacacc cagaccaggt cccctacatc gtgacctggg aagccttggc
     1201 ttttgacccc cctccctggg tcaagccctt tgtacaccct aagcctccgc ctcctcttcc
     1261 tccatccgcc ccgtctctcc cccttgaacc tcctcgttcg accccgcctc gatcctccct
     1321 ttatccagcc ctcactcctt ctctaggcgc cggccggatc agcttaccat gaccgagtac
     1381 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
     1441 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
     1501 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     1561 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
     1621 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     1681 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     1741 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     1801 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
     1861 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     1921 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgacg cccgccccac
     1981 gacccgcagc gcccgaccga aaggagcgca cgaccccatg catcggatct cccgatcccc
     2041 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc
     2101 ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc
     2161 aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc
     2221 gcgatgtacg ggccagatat acgcgttgac attgattatt gactagttat taatagtaat
     2281 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
     2341 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
     2401 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     2461 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     2521 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     2581 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
     2641 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
     2701 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
     2761 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     2821 taagcagagc tctctggcta actagagaac ccactgctta ctggcttatc gaaattaata
     2881 cgactcacta tagggagacc caagcttggt accgagctcg gatcatcaca agtttgtaca
     2941 aaaaagcagg ctccgcggcc gcccccttca ccgccaccat ggtgagcaag ggcgaggagg
     3001 ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga
     3061 acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga
     3121 ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc
     3181 ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact
     3241 tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg
     3301 gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga
     3361 agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct
     3421 gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc
     3481 agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg
     3541 ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct
     3601 cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca
     3661 ccggcggcat ggacgagctg tacaagggat ccggcggcct aaggactcgg cgcgccggaa
     3721 gtggccaggg cgggggcgac ttcggctcac agcgcgcccg gctattctcg cagctcacca
     3781 tggatgatga tatcgccgcg ctcgtcgtcg acaacggctc cggcatgtgc aaggccggct
     3841 tcagcggagc tactaacttc agcctgctga agcaggctgg agacgtggag gagaaccctg
     3901 gacctatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     3961 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     4021 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     4081 ccaccctcgt gaccaccctg acctatggag tgcagtgctt cagccgctac cccgaccaca
     4141 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     4201 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     4261 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     4321 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     4381 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     4441 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     4501 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     4561 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     4621 agtgaggatc caagggtggg cgcgccgacc cagctttctt gtacaaagtg gtgatgatcc
     4681 actagtaacg gccgccagtg tgctggaatt ctgcagatat ccatcacact ggcggccgct
     4741 cgagcatgca tctagagggc cgataaaata aaagatttta tttagtctcc agaaaaaggg
     4801 gggaatgaaa gaccccacct gtaggtttgg caagctagct taagtaacgc cattttgcaa
     4861 ggcatggaaa aatacataac tgagaataga gaagttcaga tcaaggtcag gaacagatgg
     4921 aacagctgaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc ccggctcagg
     4981 gccaagaaca gatggaacag ctgaatatgg gccaaacagg atatctgtgg taagcagttc
     5041 ctgccccggc tcagggccaa gaacagatgg tccccagatg cggtccagcc ctcagcagtt
     5101 tctagagaac catcagatgt ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt
     5161 atttgaacta accaatcagt tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc
     5221 tcaataaaag agcccacaac ccctcactcg gggcgccagt cctccgattg actgagtcgc
     5281 ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt ggtctcgctg
     5341 ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc tttcacatgc
     5401 agcatgtatc aaaattaatt tggttttttt tcttaagtat ttacattaaa tggccatagt
     5461 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     5521 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     5581 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5641 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5701 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5761 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5821 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5881 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5941 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     6001 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     6061 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     6121 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     6181 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     6241 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     6301 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     6361 gattatcaaa aaggatcttc acctagatcc ttttgcggcc gcaaatcaat ctaaagtata
     6421 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     6481 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     6541 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     6601 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     6661 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     6721 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     6781 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     6841 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     6901 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     6961 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     7021 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     7081 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     7141 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     7201 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     7261 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     7321 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     7381 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgcagc
//
