LOCUS       sequence-448187-        6226 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 6226)
  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 6226)
  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: pGEMT-PT2A-iRFP670-Tdtomato-GFP
FEATURES             Location/Qualifiers
     source          1..6226
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(29..457)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(139..160)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     351..370
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     583..605
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     597..614
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     598..614
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     621..640
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        621..639
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      693..702
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             699..1631
                     /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             1647..1703
                     /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             1710..3137
                     /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"
     CDS             3153..3206
                     /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             join(3213..3215,3216..3218,3219..3932)
                     /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(3213..3215,3216..3218,3219..3929)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3258..3279)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3519..3538)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3866..3887
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(3988..4006)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3989..4006)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4024..4040)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4024..4040)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4037..4059)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4048..4064
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4072..4078,4079..4096,4097..4102))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4117..4138
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4255..4272)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4426..5014)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4506..4525)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5185..5976,5977..6045))
                     /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     5808..5827
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6046..6150)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
       61 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
      121 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
      181 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
      241 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
      301 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
      361 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
      421 acacccgccg cgcttaatgc gccgctacag ggcgcgtcca ttcgccattc aggctgcgca
      481 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
      541 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
      601 aaacgacggc cagtgaattg taatacgact cactataggg cgaattgggc ccgacgtgat
      661 ccatcgatgt taacaccggt gacgtcggat ccgccaccat ggcgcgtaag gtcgatctca
      721 cctcctgcga tcgcgagccg atccacatcc ccggcagcat tcagccgtgc ggctgcctgc
      781 tagcctgcga cgcgcaggcg gtgcggatca cgcgcattac ggaaaatgcc ggcgcgttct
      841 ttggacgcga aactccgcgg gtcggtgagc tactcgccga ttacttcggc gagaccgaag
      901 cccatgcgct gcgcaacgca ctggcgcagt cctccgatcc aaagcgaccg gcgctgatct
      961 tcggttggcg cgacggcctg accggccgca ccttcgacat ctcactgcat cgccatgacg
     1021 gtacatcgat catcgagttc gagcctgcgg cggccgaaca ggccgacaat ccgctgcggc
     1081 tgacgcggca gatcatcgcg cgcaccaaag aactgaagtc gctcgaagag atggccgcac
     1141 gggtgccgcg ctatctgcag gcgatgctcg gctatcaccg cgtgatgttg taccgcttcg
     1201 cggacgacgg ctccgggatg gtgatcggcg aggcgaagcg cagcgacctc gagagctttc
     1261 tcggtcagca ctttccggcg tcgctggtcc cgcagcaggc gcggctactg tacttgaaga
     1321 acgcgatccg cgtggtctcg gattcgcgcg gcatcagcag ccggatcgtg cccgagcacg
     1381 acgcctccgg cgccgcgctc gatctgtcgt tcgcgcacct gcgcagcatc tcgccctgcc
     1441 atctcgaatt tctgcggaac atgggcgtca gcgcctcgat gtcgctgtcg atcatcattg
     1501 acggcacgct atggggattg atcatctgtc atcattacga gccgcgtgcc gtgccgatgg
     1561 cgcagcgcgt cgcggccgaa atgttcgccg acttcttatc gctgcacttc accgccgccc
     1621 accaccaacg cactagcggc agcggcgcca caaacttctc tctgctaaag caagcaggtg
     1681 atgttgaaga aaaccccggg cctactagta tggtgagcaa gggcgaggag gtcatcaaag
     1741 agttcatgcg cttcaaggtg cgcatggagg gctccatgaa cggccacgag ttcgagatcg
     1801 agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca
     1861 agggcggccc cctgcccttc gcctgggaca tcctgtcccc ccagttcatg tacggctcca
     1921 aggcgtacgt gaagcacccc gccgacatcc ccgattacaa gaagctgtcc ttccccgagg
     1981 gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg tctggtgacc gtgacccagg
     2041 actcctccct gcaggacggc acgctgatct acaaggtgaa gatgcgcggc accaacttcc
     2101 cccccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc accgagcgcc
     2161 tgtacccccg cgacggcgtg ctgaagggcg agatccacca ggccctgaag ctgaaggacg
     2221 gcggccacta cctggtggag ttcaagacca tctacatggc caagaagccc gtgcaactgc
     2281 ccggctacta ctacgtggac accaagctgg acatcacctc ccacaacgag gactacacca
     2341 tcgtggaaca gtacgagcgc tccgagggcc gccaccacct gttcctgggg catggcaccg
     2401 gcagcaccgg cagcggcagc tccggcaccg cctcctccga ggacaacaac atggccgtca
     2461 tcaaagagtt catgcgcttc aaggtgcgca tggagggctc catgaacggc cacgagttcg
     2521 agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg
     2581 tgaccaaggg cggccccctg cccttcgcct gggacatcct gtccccccag ttcatgtacg
     2641 gctccaaggc gtacgtgaag caccccgccg acatccccga ttacaagaag ctgtccttcc
     2701 ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggtctg gtgaccgtga
     2761 cccaggactc ctccctgcag gacggcacgc tgatctacaa ggtgaagatg cgcggcacca
     2821 acttcccccc cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctccaccg
     2881 agcgcctgta cccccgcgac ggcgtgctga agggcgagat ccaccaggcc ctgaagctga
     2941 aggacggcgg ccgctacctg gtggagttca agaccatcta catggccaag aagcccgtgc
     3001 aactgcccgg ctactactac gtggacacca agctggacat cacctcccac aacgaggact
     3061 acaccatcgt ggaacagtac gagcgctccg agggccgcca ccacctgttc ctgtacggca
     3121 tggacgagct gtacaagaag cttggctccg gcgagggcag gggaagtctt ctaacatgcg
     3181 gggacgtgga ggaaaatccc ggcccactcg agatggtgag caagggcgag gagctgttca
     3241 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     3301 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     3361 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
     3421 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     3481 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     3541 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     3601 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     3661 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
     3721 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     3781 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca
     3841 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     3901 tcactctcgg catggacgag ctgtacaagt aatctagagg agcagtcgac gcggccgccc
     3961 aacgcgttgg atgcatagct tgagtattct atagtgtcac ctaaatagct tggcgtaatc
     4021 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     4081 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     4141 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4201 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     4261 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     4321 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     4381 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4441 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4501 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4561 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     4621 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     4681 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     4741 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     4801 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     4861 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     4921 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     4981 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5041 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     5101 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     5161 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     5221 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     5281 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     5341 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     5401 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     5461 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     5521 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     5581 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     5641 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     5701 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     5761 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     5821 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     5881 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     5941 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6001 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6061 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6121 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgatgc
     6181 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcag
//
