LOCUS       sequence-214297-        7828 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 7828)
  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 7828)
  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..7828
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..594
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    657..1014
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1073..1489
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1419..1441
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1457..1473
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    1499..1873
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     1811..1827
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     regulatory      1902..1911
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1908..2840
                     /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             2856..2912
                     /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(2919..2921,2922..2924,2925..3635)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2919..2921,2922..2924,2925..3635)
                     /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(2964..2985)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3225..3244)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3572..3593
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3651..3704
                     /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             3711..5141
                     /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             5312..5781
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5911..5928)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6082..6670)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6162..6181)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6834..7625,7626..7694))
                     /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     7457..7476
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7695..7799)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc
       61 atggaaaaat acataactga gaatagaaaa gttcagatca aggtcaggaa cagatggaac
      121 agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc
      181 aagaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg
      241 ccccggctca gggccaagaa cagatggtcc ccagatgcgg tccagccctc agcagtttct
      301 agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt
      361 tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttctgctc cccgagctca
      421 ataaaagagc ccacaacccc tcactcggcg cgccagtcct ccgattgact gagtcgcccg
      481 ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc
      541 cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt catttggggg
      601 ctcgtccggg atcgggagac ccctgcccag ggaccaccga cccaccaccg ggaggtaagc
      661 tggccagcaa cttatctgtg tctgtccgat tgtctagtgt ctatgactga ttttatgcgc
      721 ctgcgtcggt actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac
      781 gagttcggaa cacccggccg caaccctggg agacgtccca gggacttcgg gggccgtttt
      841 tgtggcccga cctgagtcca aaaatcccga tcgttttgga ctctttggtg caccccccta
      901 ataggaggga tatgtggttc tggtaggaga cgagaaccta aaacagttcc cgcctccgtc
      961 tgaatttttg ctttcggttt gggaccgaag ccgcgccgcg cgtcttgtct gctgcagcat
     1021 cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaatt agggccagac
     1081 tgttaccact cccttaagtt tgaccttagg tcactggaaa gatgtcgagc ggatcgctca
     1141 caaccagtcg gtagatgtca agaagagacg ttgggttacc ttctgctctg cagaatggcc
     1201 aacctttaac gtcggatggc cgcgagacgg cacctttaac cgagacctca tcacccaggt
     1261 taagatcaag gtcttttcac ctggcccgca tggacaccca gaccaggtcc cctacatcgt
     1321 gacctgggaa gccttggctt ttgacccccc tccctgggtc aagccctttg tacaccctaa
     1381 gcctccgcct cctcttcctc catccgcccc gtctctcccc cttgaacctc ctcgttcgac
     1441 cccgcctcga tcctcccttt atccagccct cactccttct ctaggcgccc ccatatggcc
     1501 atatgagatc ttatatgggg cacccccgcc ccttgtaaac ttccctgacc ctgacatgac
     1561 aagagttact aacagcccct ctctccaagc tcacttacag gctctctact tagtccagca
     1621 cgaagtctgg agacctctgg cggcagccta ccaagaacaa ctggaccgac cggtggtacc
     1681 tcacccttac cgagtcggcg acacagtgtg ggtccgccga caccagacta agaacctaga
     1741 acctcgctgg aaaggacctt acacagtcct gctgaccacc cccaccgccc tcaaagtaga
     1801 cggcatcgca gcttggatac acgccgccca cgtgaaggct gccgaccccg ggggtggacc
     1861 atcctctaga ctgccggatc tagctagtta attaaggatc cgccaccatg gcgcgtaagg
     1921 tcgatctcac ctcctgcgat cgcgagccga tccacatccc cggcagcatt cagccgtgcg
     1981 gctgcctgct agcctgcgac gcgcaggcgg tgcggatcac gcgcattacg gaaaatgccg
     2041 gcgcgttctt tggacgcgaa actccgcggg tcggtgagct actcgccgat tacttcggcg
     2101 agaccgaagc ccatgcgctg cgcaacgcac tggcgcagtc ctccgatcca aagcgaccgg
     2161 cgctgatctt cggttggcgc gacggcctga ccggccgcac cttcgacatc tcactgcatc
     2221 gccatgacgg tacatcgatc atcgagttcg agcctgcggc ggccgaacag gccgacaatc
     2281 cgctgcggct gacgcggcag atcatcgcgc gcaccaaaga actgaagtcg ctcgaagaga
     2341 tggccgcacg ggtgccgcgc tatctgcagg cgatgctcgg ctatcaccgc gtgatgttgt
     2401 accgcttcgc ggacgacggc tccgggatgg tgatcggcga ggcgaagcgc agcgacctcg
     2461 agagctttct cggtcagcac tttccggcgt cgctggtccc gcagcaggcg cggctactgt
     2521 acttgaagaa cgcgatccgc gtggtctcgg attcgcgcgg catcagcagc cggatcgtgc
     2581 ccgagcacga cgcctccggc gccgcgctcg atctgtcgtt cgcgcacctg cgcagcatct
     2641 cgccctgcca tctcgaattt ctgcggaaca tgggcgtcag cgcctcgatg tcgctgtcga
     2701 tcatcattga cggcacgcta tggggattga tcatctgtca tcattacgag ccgcgtgccg
     2761 tgccgatggc gcagcgcgtc gcggccgaaa tgttcgccga cttcttatcg ctgcacttca
     2821 ccgccgccca ccaccaacgc actagcggca gcggcgccac aaacttctct ctgctaaagc
     2881 aagcaggtga tgttgaagaa aaccccgggc ctactagtat ggtgagcaag ggcgaggagc
     2941 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     3001 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     3061 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
     3121 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     3181 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     3241 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     3301 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     3361 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
     3421 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     3481 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc
     3541 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     3601 ccgggatcac tctcggcatg gacgagctgt acaagaagct tggctccggc gagggcaggg
     3661 gaagtcttct aacatgcggg gacgtggagg aaaatcccgg cccactcgag atggtgagca
     3721 agggcgagga ggtcatcaaa gagttcatgc gcttcaaggt gcgcatggag ggctccatga
     3781 acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga
     3841 ccgccaagct gaaggtgacc aagggcggcc ccctgccctt cgcctgggac atcctgtccc
     3901 cccagttcat gtacggctcc aaggcgtacg tgaagcaccc cgccgacatc cccgattaca
     3961 agaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg
     4021 gtctggtgac cgtgacccag gactcctccc tgcaggacgg cacgctgatc tacaaggtga
     4081 agatgcgcgg caccaacttc ccccccgacg gccccgtaat gcagaagaag accatgggct
     4141 gggaggcctc caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc gagatccacc
     4201 aggccctgaa gctgaaggac ggcggccact acctggtgga gttcaagacc atctacatgg
     4261 ccaagaagcc cgtgcaactg cccggctact actacgtgga caccaagctg gacatcacct
     4321 cccacaacga ggactacacc atcgtggaac agtacgagcg ctccgagggc cgccaccacc
     4381 tgttcctggg gcatggcacc ggcagcaccg gcagcggcag ctccggcacc gcctcctccg
     4441 aggacaacaa catggccgtc atcaaagagt tcatgcgctt caaggtgcgc atggagggct
     4501 ccatgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca
     4561 cccagaccgc caagctgaag gtgaccaagg gcggccccct gcccttcgcc tgggacatcc
     4621 tgtcccccca gttcatgtac ggctccaagg cgtacgtgaa gcaccccgcc gacatccccg
     4681 attacaagaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg
     4741 acggcggtct ggtgaccgtg acccaggact cctccctgca ggacggcacg ctgatctaca
     4801 aggtgaagat gcgcggcacc aacttccccc ccgacggccc cgtaatgcag aagaagacca
     4861 tgggctggga ggcctccacc gagcgcctgt acccccgcga cggcgtgctg aagggcgaga
     4921 tccaccaggc cctgaagctg aaggacggcg gccgctacct ggtggagttc aagaccatct
     4981 acatggccaa gaagcccgtg caactgcccg gctactacta cgtggacacc aagctggaca
     5041 tcacctccca caacgaggac tacaccatcg tggaacagta cgagcgctcc gagggccgcc
     5101 accacctgtt cctgtacggc atggacgagc tgtacaagta agtcgacgat aaaataaaag
     5161 attttattta gtctccagaa aaagggggga atgaaagacc ccacctgtag gtttggcaag
     5221 ctagcttaag taacgccatt ttgcaaggca tggaaaaata cataactgag aatagagaag
     5281 ttcagatcaa ggtcaggaac agatggaaca gctgaatatg ggccaaacag gatatctgtg
     5341 gtaagcagtt cctgccccgg ctcagggcca agaacagatg gaacagctga atatgggcca
     5401 aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggtccc
     5461 cagatgcggt ccagccctca gcagtttcta gagaaccatc agatgtttcc agggtgcccc
     5521 aaggacctga aatgaccctg tgccttattt gaactaacca atcagttcgc ttctcgcttc
     5581 tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc cacaacccct cactcggggc
     5641 gccagtcctc cgattgactg agtcgcccgg gtacccgtgt atccaataaa ccctcttgca
     5701 gttgcatccg acttgtggtc tcgctgttcc ttgggagggt ctcctctgag tgattgacta
     5761 cccgtcagcg ggggtctttc acatgcagca tgtatcaaaa ttaatttggt tttttttctt
     5821 aagtatttac attaaatggc catagttgca ttaatgaatc ggccaacgcg cggggagagg
     5881 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     5941 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     6001 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     6061 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     6121 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     6181 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     6241 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     6301 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     6361 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     6421 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     6481 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     6541 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     6601 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     6661 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     6721 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     6781 gcggccggcc gcaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6841 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     6901 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     6961 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     7021 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     7081 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     7141 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     7201 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     7261 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     7321 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     7381 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     7441 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     7501 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     7561 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     7621 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     7681 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     7741 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     7801 acatttcccc gaaaagtgcc acctgcat
//
