LOCUS       sequence-320919-        7439 bp    DNA     circular SYN 14-APR-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 Apr 14, 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"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    681..705
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      726..735
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             732..1439
                     /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(877..895)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1145..1164
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1602..1658
                     /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(1659..1661,1662..1664,1665..2378)
                     /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(1659..1661,1662..1664,1665..2375)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1704..1725)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1965..1984)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2312..2333
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2401..2425)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     LTR             2679..3148
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(3278..3295)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3449..4037)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3529..3548)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4197..4988,4989..5057))
                     /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     4820..4839
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5058..5162)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             5193..5662
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    5727..5926
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             6127..6543
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     6473..6495
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     6511..6527
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     CDS             6561..7160
                     /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(6561..6580)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7057..7077
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(7249..7268)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 ggtaccgagc tcggatcatc acaagtttgt acaaaaaagc aggctccgcg gccgccccct
      721 tcaccgccac catggtgagc aagggcgagg aggataacat ggccatcatc aaggagttca
      781 tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg
      841 agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg
      901 gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct
      961 acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca
     1021 agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct
     1081 ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg
     1141 acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag cggatgtacc
     1201 ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc
     1261 actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg
     1321 cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg
     1381 aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagg
     1441 gatccggcgg cctaaggact cggcgcgccg gaagtggcca gggcgggggc gacttcggct
     1501 cacagcgcgc ccggctattc tcgcagctca ccatggatga tgatatcgcc gcgctcgtcg
     1561 tcgacaacgg ctccggcatg tgcaaggccg gcttcagcgg agctactaac ttcagcctgc
     1621 tgaagcaggc tggagacgtg gaggagaacc ctggacctat ggtgagcaag ggcgaggagc
     1681 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     1741 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     1801 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctatg
     1861 gagtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     1921 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     1981 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     2041 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     2101 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
     2161 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     2221 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc
     2281 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     2341 ccgggatcac tctcggcatg gacgagctgt acaagtgagg atccaagggt gggcgcgccg
     2401 acccagcttt cttgtacaaa gtggtgatga tccactagta acggccgcca gtgtgctgga
     2461 attctgcaga tatccatcac actggcggcc gctcgagcat gcatctagag ggccgataaa
     2521 ataaaagatt ttatttagtc tccagaaaaa ggggggaatg aaagacccca cctgtaggtt
     2581 tggcaagcta gcttaagtaa cgccattttg caaggcatgg aaaaatacat aactgagaat
     2641 agagaagttc agatcaaggt caggaacaga tggaacagct gaatatgggc caaacaggat
     2701 atctgtggta agcagttcct gccccggctc agggccaaga acagatggaa cagctgaata
     2761 tgggccaaac aggatatctg tggtaagcag ttcctgcccc ggctcagggc caagaacaga
     2821 tggtccccag atgcggtcca gccctcagca gtttctagag aaccatcaga tgtttccagg
     2881 gtgccccaag gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc
     2941 tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa aagagcccac aacccctcac
     3001 tcggggcgcc agtcctccga ttgactgagt cgcccgggta cccgtgtatc caataaaccc
     3061 tcttgcagtt gcatccgact tgtggtctcg ctgttccttg ggagggtctc ctctgagtga
     3121 ttgactaccc gtcagcgggg gtctttcaca tgcagcatgt atcaaaatta atttggtttt
     3181 ttttcttaag tatttacatt aaatggccat agttgcatta atgaatcggc caacgcgcgg
     3241 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     3301 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     3361 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     3421 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     3481 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     3541 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     3601 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     3661 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     3721 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     3781 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     3841 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     3901 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     3961 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4021 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4081 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4141 tccttttgcg gccgcaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4201 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4261 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     4321 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     4381 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     4441 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     4501 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     4561 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     4621 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     4681 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     4741 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     4801 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     4861 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     4921 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     4981 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     5041 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     5101 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5161 cgcacatttc cccgaaaagt gccacctgca gcctgaatat gggccaaaca ggatatctgt
     5221 ggtaagcagt tcctgccccg gctcagggcc aagaacagat ggaacagctg aatatgggcc
     5281 aaacaggata tctgtggtaa gcagttcctg ccccggctca gggccaagaa cagatggtcc
     5341 ccagatgcgg tccagccctc agcagtttct agagaaccat cagatgtttc cagggtgccc
     5401 caaggacctg aaatgaccct gtgccttatt tgaactaacc aatcagttcg cttctcgctt
     5461 ctgttcgcgc gcttctgctc cccgagctca ataaaagagc ccacaacccc tcactcgggg
     5521 cgccagtcct ccgattgact gagtcgcccg ggtacccgtg tatccaataa accctcttgc
     5581 agttgcatcc gacttgtggt ctcgctgttc cttgggaggg tctcctctga gtgattgact
     5641 acccgtcagc gggggtcttt catttggggg ctcgtccggg atcgggagac ccctgcccag
     5701 ggaccaccga cccaccaccg ggaggcaagc tggccagcaa cttatctgtg tctgtccgat
     5761 tgtctagtgt ctatgactga ttttatgcgc ctgcgtcggt actagttagc taactagctc
     5821 tgtatctggc ggacccgtgg tggaactgac gagttctgaa cacccggccg caaccctggg
     5881 agacgtccca gggactttgg gggccgtttt tgtggcccga cctgaggaag ggagtcgatg
     5941 tggaatccga ccccgtcagg atatgtggtt ctggtaggag acgagaacct aaaacagttc
     6001 ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa gccgcgcgtc ttgtctgctg
     6061 cagcgctgca gcatcgttct gtgttgtctc tgtctgactg tgtttctgta tttgtctgaa
     6121 aattagggcc agactgttac cactccctta agtttgacct taggtcactg gaaagatgtc
     6181 gagcggatcg ctcacaacca gtcggtagat gtcaagaaga gacgttgggt taccttctgc
     6241 tctgcagaat ggccaacctt taacgtcgga tggccgcgag acggcacctt taaccgagac
     6301 ctcatcaccc aggttaagat caaggtcttt tcacctggcc cgcatggaca cccagaccag
     6361 gtcccctaca tcgtgacctg ggaagccttg gcttttgacc cccctccctg ggtcaagccc
     6421 tttgtacacc ctaagcctcc gcctcctctt cctccatccg ccccgtctct cccccttgaa
     6481 cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag ccctcactcc ttctctaggc
     6541 gccggccgga tcagcttacc atgaccgagt acaagcccac ggtgcgcctc gccacccgcg
     6601 acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc
     6661 gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc
     6721 tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg
     6781 cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc
     6841 gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg
     6901 cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc
     6961 accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg
     7021 ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc
     7081 tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga
     7141 cccgcaagcc cggtgcctga cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg
     7201 cacgacccca tgcatcggat ctcccgatcc cctatggtgc actctcagta caatctgctc
     7261 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta
     7321 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa
     7381 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgtt
//
