LOCUS       sequence-210072-        7827 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tricistronic construct: Co-express three fluorescent proteins by P2A
            and then T2A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7827)
  AUTHORS   Liu Z, Chen O, Wall JBJ, Zheng M, Zhou Y, Wang L, Ruth Vaseghi H, 
            Qian L, Liu J
  TITLE     Systematic comparison of 2A peptides for cloning multi-genes in a 
            polycistronic vector.
  JOURNAL   Sci Rep. 2017 May 19;7(1):2193. doi: 10.1038/s41598-017-02460-2.
  PUBMED    28526819
REFERENCE   2  (bases 1 to 7827)
  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: "Sci Rep."; 
            date: "2017-05-19"; volume: "7(1):2193. doi"; pages: " 
            10.1038/s41598-017-02460-2"
COMMENT     Sequence Label: pMXs-PT2A-iRFP670-GFP-Tdtomato
FEATURES             Location/Qualifiers
     source          1..7827
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(2..21)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(674..1465,1466..1534))
                     /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     1297..1316
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1535..1639)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             1669..2262
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    2325..2682
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             2741..3157
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     3087..3109
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     3125..3141
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    3167..3541
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     3479..3495
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     regulatory      3570..3579
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3576..4508
                     /codon_start=1
                     /product="near-infrared fluorescent protein with an 
                     emission peak at 670 nm, engineered from a bacterial 
                     phytochrome (Shcherbakova and Verkhusha, 2013)"
                     /label=iRFP670
                     /note="derived from Rhodopseudomonas palustris RpBphP6"
                     /translation="MARKVDLTSCDREPIHIPGSIQPCGCLLACDAQAVRITRITENAG
                     AFFGRETPRVGELLADYFGETEAHALRNALAQSSDPKRPALIFGWRDGLTGRTFDISLH
                     RHDGTSIIEFEPAAAEQADNPLRLTRQIIARTKELKSLEEMAARVPRYLQAMLGYHRVM
                     LYRFADDGSGMVIGEAKRSDLESFLGQHFPASLVPQQARLLYLKNAIRVVSDSRGISSR
                     IVPEHDASGAALDLSFAHLRSISPCHLEFLRNMGVSASMSLSIIIDGTLWGLIICHHYE
                     PRAVPMAQRVAAEMFADFLSLHFTAAHHQR"
     CDS             4524..4580
                     /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(4587..4589,4590..4592,4593..5303)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4587..4589,4590..4592,4593..5303)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4632..4653)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4893..4912)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5240..5261
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5319..5372
                     /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             5379..6809
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGRYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     LTR             6980..7449
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(7578..7595)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(7749..7827,1..510))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
       61 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
      121 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
      181 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
      241 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
      301 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
      361 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
      421 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
      481 cagattacgc gcagaaaaaa nggatctcaa gaagatcctt tgatcttttc tacggggtct
      541 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
      601 atcttcacct agatcctttt gcggccggcc gcaaatcaat ctaaagtata tatgagtaaa
      661 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
      721 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
      781 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
      841 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
      901 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
      961 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     1021 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     1081 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     1141 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     1201 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     1261 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     1321 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     1381 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     1441 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     1501 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     1561 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     1621 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgcataa tgaaagaccc
     1681 cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat ggaaaaatac
     1741 ataactgaga atagaaaagt tcagatcaag gtcaggaaca gatggaacag ctgaatatgg
     1801 gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg
     1861 aacagctgaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc ccggctcagg
     1921 gccaagaaca gatggtcccc agatgcggtc cagccctcag cagtttctag agaaccatca
     1981 gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg aactaaccaa
     2041 tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc
     2101 acaacccctc actcggcgcg ccagtcctcc gattgactga gtcgcccggg tacccgtgta
     2161 tccaataaac cctcttgcag ttgcatccga cttgtggtct cgctgttcct tgggagggtc
     2221 tcctctgagt gattgactac ccgtcagcgg gggtctttca tttgggggct cgtccgggat
     2281 cgggagaccc ctgcccaggg accaccgacc caccaccggg aggtaagctg gccagcaact
     2341 tatctgtgtc tgtccgattg tctagtgtct atgactgatt ttatgcgcct gcgtcggtac
     2401 tagttagcta actagctctg tatctggcgg acccgtggtg gaactgacga gttcggaaca
     2461 cccggccgca accctgggag acgtcccagg gacttcgggg gccgtttttg tggcccgacc
     2521 tgagtccaaa aatcccgatc gttttggact ctttggtgca cccccctaat aggagggata
     2581 tgtggttctg gtaggagacg agaacctaaa acagttcccg cctccgtctg aatttttgct
     2641 ttcggtttgg gaccgaagcc gcgccgcgcg tcttgtctgc tgcagcatcg ttctgtgttg
     2701 tctctgtctg actgtgtttc tgtatttgtc tgaaaattag ggccagactg ttaccactcc
     2761 cttaagtttg accttaggtc actggaaaga tgtcgagcgg atcgctcaca accagtcggt
     2821 agatgtcaag aagagacgtt gggttacctt ctgctctgca gaatggccaa cctttaacgt
     2881 cggatggccg cgagacggca cctttaaccg agacctcatc acccaggtta agatcaaggt
     2941 cttttcacct ggcccgcatg gacacccaga ccaggtcccc tacatcgtga cctgggaagc
     3001 cttggctttt gacccccctc cctgggtcaa gccctttgta caccctaagc ctccgcctcc
     3061 tcttcctcca tccgccccgt ctctccccct tgaacctcct cgttcgaccc cgcctcgatc
     3121 ctccctttat ccagccctca ctccttctct aggcgccccc atatggccat atgagatctt
     3181 atatggggca cccccgcccc ttgtaaactt ccctgaccct gacatgacaa gagttactaa
     3241 cagcccctct ctccaagctc acttacaggc tctctactta gtccagcacg aagtctggag
     3301 acctctggcg gcagcctacc aagaacaact ggaccgaccg gtggtacctc acccttaccg
     3361 agtcggcgac acagtgtggg tccgccgaca ccagactaag aacctagaac ctcgctggaa
     3421 aggaccttac acagtcctgc tgaccacccc caccgccctc aaagtagacg gcatcgcagc
     3481 ttggatacac gccgcccacg tgaaggctgc cgaccccggg ggtggaccat cctctagact
     3541 gccggatcta gctagttaat taaggatccg ccaccatggc gcgtaaggtc gatctcacct
     3601 cctgcgatcg cgagccgatc cacatccccg gcagcattca gccgtgcggc tgcctgctag
     3661 cctgcgacgc gcaggcggtg cggatcacgc gcattacgga aaatgccggc gcgttctttg
     3721 gacgcgaaac tccgcgggtc ggtgagctac tcgccgatta cttcggcgag accgaagccc
     3781 atgcgctgcg caacgcactg gcgcagtcct ccgatccaaa gcgaccggcg ctgatcttcg
     3841 gttggcgcga cggcctgacc ggccgcacct tcgacatctc actgcatcgc catgacggta
     3901 catcgatcat cgagttcgag cctgcggcgg ccgaacaggc cgacaatccg ctgcggctga
     3961 cgcggcagat catcgcgcgc accaaagaac tgaagtcgct cgaagagatg gccgcacggg
     4021 tgccgcgcta tctgcaggcg atgctcggct atcaccgcgt gatgttgtac cgcttcgcgg
     4081 acgacggctc cgggatggtg atcggcgagg cgaagcgcag cgacctcgag agctttctcg
     4141 gtcagcactt tccggcgtcg ctggtcccgc agcaggcgcg gctactgtac ttgaagaacg
     4201 cgatccgcgt ggtctcggat tcgcgcggca tcagcagccg gatcgtgccc gagcacgacg
     4261 cctccggcgc cgcgctcgat ctgtcgttcg cgcacctgcg cagcatctcg ccctgccatc
     4321 tcgaatttct gcggaacatg ggcgtcagcg cctcgatgtc gctgtcgatc atcattgacg
     4381 gcacgctatg gggattgatc atctgtcatc attacgagcc gcgtgccgtg ccgatggcgc
     4441 agcgcgtcgc ggccgaaatg ttcgccgact tcttatcgct gcacttcacc gccgcccacc
     4501 accaacgcgc tagcggcagc ggcgccacaa acttctctct gctaaagcaa gcaggtgatg
     4561 ttgaagaaaa ccccgggcct actagtatgg tgagcaaggg cgaggagctg ttcaccgggg
     4621 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     4681 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     4741 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     4801 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     4861 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     4921 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     4981 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     5041 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     5101 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     5161 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     5221 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     5281 tcggcatgga cgagctgtac aagaagcttg gctccggcga gggcagggga agtcttctaa
     5341 catgcgggga cgtggaggaa aatcccggcc cactcgagat ggtgagcaag ggcgaggagg
     5401 tcatcaaaga gttcatgcgc ttcaaggtgc gcatggaggg ctccatgaac ggccacgagt
     5461 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga
     5521 aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtccccc cagttcatgt
     5581 acggctccaa ggcgtacgtg aagcaccccg ccgacatccc cgattacaag aagctgtcct
     5641 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggt ctggtgaccg
     5701 tgacccagga ctcctccctg caggacggca cgctgatcta caaggtgaag atgcgcggca
     5761 ccaacttccc ccccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcca
     5821 ccgagcgcct gtacccccgc gacggcgtgc tgaagggcga gatccaccag gccctgaagc
     5881 tgaaggacgg cggccactac ctggtggagt tcaagaccat ctacatggcc aagaagcccg
     5941 tgcaactgcc cggctactac tacgtggaca ccaagctgga catcacctcc cacaacgagg
     6001 actacaccat cgtggaacag tacgagcgct ccgagggccg ccaccacctg ttcctggggc
     6061 atggcaccgg cagcaccggc agcggcagct ccggcaccgc ctcctccgag gacaacaaca
     6121 tggccgtcat caaagagttc atgcgcttca aggtgcgcat ggagggctcc atgaacggcc
     6181 acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca
     6241 agctgaaggt gaccaagggc ggccccctgc ccttcgcctg ggacatcctg tccccccagt
     6301 tcatgtacgg ctccaaggcg tacgtgaagc accccgccga catccccgat tacaagaagc
     6361 tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggtctgg
     6421 tgaccgtgac ccaggactcc tccctgcagg acggcacgct gatctacaag gtgaagatgc
     6481 gcggcaccaa cttccccccc gacggccccg taatgcagaa gaagaccatg ggctgggagg
     6541 cctccaccga gcgcctgtac ccccgcgacg gcgtgctgaa gggcgagatc caccaggccc
     6601 tgaagctgaa ggacggcggc cgctacctgg tggagttcaa gaccatctac atggccaaga
     6661 agcccgtgca actgcccggc tactactacg tggacaccaa gctggacatc acctcccaca
     6721 acgaggacta caccatcgtg gaacagtacg agcgctccga gggccgccac cacctgttcc
     6781 tgtacggcat ggacgagctg tacaagtaag tcgacgataa aataaaagat tttatttagt
     6841 ctccagaaaa aggggggaat gaaagacccc acctgtaggt ttggcaagct agcttaagta
     6901 acgccatttt gcaaggcatg gaaaaataca taactgagaa tagagaagtt cagatcaagg
     6961 tcaggaacag atggaacagc tgaatatggg ccaaacagga tatctgtggt aagcagttcc
     7021 tgccccggct cagggccaag aacagatgga acagctgaat atgggccaaa caggatatct
     7081 gtggtaagca gttcctgccc cggctcaggg ccaagaacag atggtcccca gatgcggtcc
     7141 agccctcagc agtttctaga gaaccatcag atgtttccag ggtgccccaa ggacctgaaa
     7201 tgaccctgtg ccttatttga actaaccaat cagttcgctt ctcgcttctg ttcgcgcgct
     7261 tctgctcccc gagctcaata aaagagccca caacccctca ctcggggcgc cagtcctccg
     7321 attgactgag tcgcccgggt acccgtgtat ccaataaacc ctcttgcagt tgcatccgac
     7381 ttgtggtctc gctgttcctt gggagggtct cctctgagtg attgactacc cgtcagcggg
     7441 ggtctttcac atgcagcatg tatcaaaatt aatttggttt ttttcttaag tatttacatt
     7501 aaatggccat agttgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     7561 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     7621 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     7681 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     7741 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     7801 cagaggtggc gaaacccgac aggacta
//
