LOCUS       sequence-214540-        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-GFP-Tdtomato-iRFP670
FEATURES             Location/Qualifiers
     source          1..6226
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     24..43
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        24..42
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      96..105
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(102..104,105..107,108..818)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(102..104,105..107,108..818)
                     /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(147..168)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(408..427)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     755..776
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             834..890
                     /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             897..2324
                     /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             2340..2393
                     /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             2400..3335
                     /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"
     promoter        complement(3391..3409)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3392..3409)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3427..3443)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3427..3443)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3440..3462)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3451..3467
                     /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(3475..3481,3482..3499,3500..3505))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3520..3541
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3658..3675)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3829..4417)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3909..3928)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4588..5379,5380..5448))
                     /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     5211..5230
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5449..5553)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5631..6086)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5768..5789)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5980..5999
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     join(6212..6226,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(6226,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattg ggcccgacgt
       61 gatccatcga tgttaacacc ggtgacgtcg gatccgccac catggtgagc aagggcgagg
      121 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
      181 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
      241 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
      301 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
      361 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
      421 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
      481 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
      541 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
      601 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
      661 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
      721 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
      781 ccgccgggat cactctcggc atggacgagc tgtacaaggc tagcggcagc ggcgccacaa
      841 acttctctct gctaaagcaa gcaggtgatg ttgaagaaaa ccccgggcct actagtatgg
      901 tgagcaaggg cgaggaggtc atcaaagagt tcatgcgctt caaggtgcgc atggagggct
      961 ccatgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca
     1021 cccagaccgc caagctgaag gtgaccaagg gcggccccct gcccttcgcc tgggacatcc
     1081 tgtcccccca gttcatgtac ggctccaagg cgtacgtgaa gcaccccgcc gacatccccg
     1141 attacaagaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg
     1201 acggcggtct ggtgaccgtg acccaggact cctccctgca ggacggcacg ctgatctaca
     1261 aggtgaagat gcgcggcacc aacttccccc ccgacggccc cgtaatgcag aagaagacca
     1321 tgggctggga ggcctccacc gagcgcctgt acccccgcga cggcgtgctg aagggcgaga
     1381 tccaccaggc cctgaagctg aaggacggcg gccactacct ggtggagttc aagaccatct
     1441 acatggccaa gaagcccgtg caactgcccg gctactacta cgtggacacc aagctggaca
     1501 tcacctccca caacgaggac tacaccatcg tggaacagta cgagcgctcc gagggccgcc
     1561 accacctgtt cctggggcat ggcaccggca gcaccggcag cggcagctcc ggcaccgcct
     1621 cctccgagga caacaacatg gccgtcatca aagagttcat gcgcttcaag gtgcgcatgg
     1681 agggctccat gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
     1741 agggcaccca gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg
     1801 acatcctgtc cccccagttc atgtacggct ccaaggcgta cgtgaagcac cccgccgaca
     1861 tccccgatta caagaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact
     1921 tcgaggacgg cggtctggtg accgtgaccc aggactcctc cctgcaggac ggcacgctga
     1981 tctacaaggt gaagatgcgc ggcaccaact tcccccccga cggccccgta atgcagaaga
     2041 agaccatggg ctgggaggcc tccaccgagc gcctgtaccc ccgcgacggc gtgctgaagg
     2101 gcgagatcca ccaggccctg aagctgaagg acggcggccg ctacctggtg gagttcaaga
     2161 ccatctacat ggccaagaag cccgtgcaac tgcccggcta ctactacgtg gacaccaagc
     2221 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgag cgctccgagg
     2281 gccgccacca cctgttcctg tacggcatgg acgagctgta caagaagctt ggctccggcg
     2341 agggcagggg aagtcttcta acatgcgggg acgtggagga aaatcccggc ccactcgaca
     2401 tggcgcgtaa ggtcgatctc acctcctgcg atcgcgagcc gatccacatc cccggcagca
     2461 ttcagccgtg cggctgcctg ctagcctgcg acgcgcaggc ggtgcggatc acgcgcatta
     2521 cggaaaatgc cggcgcgttc tttggacgcg aaactccgcg ggtcggtgag ctactcgccg
     2581 attacttcgg cgagaccgaa gcccatgcgc tgcgcaacgc actggcgcag tcctccgatc
     2641 caaagcgacc ggcgctgatc ttcggttggc gcgacggcct gaccggccgc accttcgaca
     2701 tctcactgca tcgccatgac ggtacatcga tcatcgagtt cgagcctgcg gcggccgaac
     2761 aggccgacaa tccgctgcgg ctgacgcggc agatcatcgc gcgcaccaaa gaactgaagt
     2821 cgctcgaaga gatggccgca cgggtgccgc gctatctgca ggcgatgctc ggctatcacc
     2881 gcgtgatgtt gtaccgcttc gcggacgacg gctccgggat ggtgatcggc gaggcgaagc
     2941 gcagcgacct cgagagcttt ctcggtcagc actttccggc gtcgctggtc ccgcagcagg
     3001 cgcggctact gtacttgaag aacgcgatcc gcgtggtctc ggattcgcgc ggcatcagca
     3061 gccggatcgt gcccgagcac gacgcctccg gcgccgcgct cgatctgtcg ttcgcgcacc
     3121 tgcgcagcat ctcgccctgc catctcgaat ttctgcggaa catgggcgtc agcgcctcga
     3181 tgtcgctgtc gatcatcatt gacggcacgc tatggggatt gatcatctgt catcattacg
     3241 agccgcgtgc cgtgccgatg gcgcagcgcg tcgcggccga aatgttcgcc gacttcttat
     3301 cgctgcactt caccgccgcc caccaccaac gctaatctag aggagcagtc gacgcggccg
     3361 cccaacgcgt tggatgcata gcttgagtat tctatagtgt cacctaaata gcttggcgta
     3421 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     3481 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     3541 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     3601 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     3661 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     3721 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     3781 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     3841 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     3901 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     3961 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     4021 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     4081 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     4141 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     4201 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     4261 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     4321 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     4381 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4441 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     4501 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     4561 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     4621 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     4681 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     4741 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     4801 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     4861 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     4921 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     4981 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     5041 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     5101 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     5161 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     5221 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     5281 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     5341 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     5401 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     5461 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     5521 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     5581 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg aaattgtaag
     5641 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     5701 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     5761 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     5821 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     5881 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     5941 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     6001 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     6061 gcttaatgcg ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa ctgttgggaa
     6121 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca
     6181 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgtt
//
