LOCUS       sequence-160915-        4790 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes P2A-mCherry preceded by a multiple cloning sequence. Enables
            bicistronic expression of protein of interest and mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4790)
  AUTHORS   van Unen J, Rashidfarrokhi A, Hoogendoorn E, Postma M, Gadella TW 
            Jr, Goedhart J
  TITLE     Quantitative Single-Cell Analysis of Signaling Pathways Activated 
            Immediately Downstream of Histamine Receptor Subtypes.
  JOURNAL   Mol Pharmacol. 2016 Sep;90(3):162-76. doi: 10.1124/mol.116.104505. 
            Epub 2016 Jun 29.
  PUBMED    27358232
REFERENCE   2  (bases 1 to 4790)
  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: 
            "10.1124/mol.116.104505"; journalName: "Mol Pharmacol"; date: 
            "2016-09"; volume: "90"; issue: "3"; pages: "162-76"
COMMENT     Sequence Label: pP2A-mCherry-N1
FEATURES             Location/Qualifiers
     source          1..4790
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    591..671
                     /label=MCS
                     /note="multiple cloning site"
     CDS             682..738
                     /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             745..1455
                     /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(890..908)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1158..1177
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    1578..1699
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1615..1634)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1669..1688
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1706..2161)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1843..1864)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2055..2074
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2188..2292
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2294..2651
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2294..2485
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2317..2337)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2502..2637
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2686..3480
                     /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             2686..3480
                     /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(2740..2759)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3350..3369
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3668..3687)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3712..3759
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4088..4676
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4577..4596
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg
      661 gatccaccgg tgggatcggg tgctactaac ttcagcctgc tgaagcaggc tggagacgtg
      721 gaggagaacc ctggacctgg gtccatggtg agcaagggcg aggaggataa catggccatc
      781 atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc
      841 gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag
      901 gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac
      961 ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc
     1021 cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg
     1081 acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc
     1141 aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc
     1201 gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg
     1261 aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg
     1321 cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac
     1381 tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac
     1441 gagctgtaca agtaaagcgg ccgcgactct agatcataat cagccatacc acatttgtag
     1501 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga
     1561 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     1621 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     1681 aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg
     1741 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     1801 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     1861 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     1921 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     1981 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     2041 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     2101 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     2161 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     2221 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     2281 aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg
     2341 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     2401 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     2461 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     2521 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     2581 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     2641 gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     2701 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     2761 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     2821 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg
     2881 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     2941 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     3001 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     3061 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     3121 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     3181 gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc
     3241 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     3301 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     3361 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     3421 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     3481 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     3541 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     3601 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga
     3661 ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa
     3721 aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc
     3781 agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt
     3841 cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg
     3901 tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag attgatttaa
     3961 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     4021 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     4081 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     4141 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     4201 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     4261 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     4321 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     4381 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     4441 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     4501 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     4561 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     4621 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     4681 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     4741 ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatgcat
//
