LOCUS       sequence-231535-        6198 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 6198)
  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 6198)
  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-Tdtomato-iRFP670-GFP
FEATURES             Location/Qualifiers
     source          1..6198
                     /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      68..77
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             74..1501
                     /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             1517..1573
                     /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             1580..2512
                     /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             2528..2581
                     /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(2588..2590,2591..2593,2594..3307)
                     /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(2588..2590,2591..2593,2594..3304)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2633..2654)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2894..2913)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3241..3262
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(3363..3381)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3364..3381)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3399..3415)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3399..3415)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3412..3434)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3423..3439
                     /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(3447..3453,3454..3471,3472..3477))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3492..3513
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3630..3647)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3801..4389)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3881..3900)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4560..5351,5352..5420))
                     /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     5183..5202
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5421..5525)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5603..6058)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5740..5761)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5952..5971
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     join(6184..6198,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(6198,1..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 cggatccgcc accatggtga gcaagggcga ggaggtcatc aaagagttca tgcgcttcaa
      121 ggtgcgcatg gagggctcca tgaacggcca cgagttcgag atcgagggcg agggcgaggg
      181 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggcg gccccctgcc
      241 cttcgcctgg gacatcctgt ccccccagtt catgtacggc tccaaggcgt acgtgaagca
      301 ccccgccgac atccccgatt acaagaagct gtccttcccc gagggcttca agtgggagcg
      361 cgtgatgaac ttcgaggacg gcggtctggt gaccgtgacc caggactcct ccctgcagga
      421 cggcacgctg atctacaagg tgaagatgcg cggcaccaac ttcccccccg acggccccgt
      481 aatgcagaag aagaccatgg gctgggaggc ctccaccgag cgcctgtacc cccgcgacgg
      541 cgtgctgaag ggcgagatcc accaggccct gaagctgaag gacggcggcc actacctggt
      601 ggagttcaag accatctaca tggccaagaa gcccgtgcaa ctgcccggct actactacgt
      661 ggacaccaag ctggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga
      721 gcgctccgag ggccgccacc acctgttcct ggggcatggc accggcagca ccggcagcgg
      781 cagctccggc accgcctcct ccgaggacaa caacatggcc gtcatcaaag agttcatgcg
      841 cttcaaggtg cgcatggagg gctccatgaa cggccacgag ttcgagatcg agggcgaggg
      901 cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca agggcggccc
      961 cctgcccttc gcctgggaca tcctgtcccc ccagttcatg tacggctcca aggcgtacgt
     1021 gaagcacccc gccgacatcc ccgattacaa gaagctgtcc ttccccgagg gcttcaagtg
     1081 ggagcgcgtg atgaacttcg aggacggcgg tctggtgacc gtgacccagg actcctccct
     1141 gcaggacggc acgctgatct acaaggtgaa gatgcgcggc accaacttcc cccccgacgg
     1201 ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc accgagcgcc tgtacccccg
     1261 cgacggcgtg ctgaagggcg agatccacca ggccctgaag ctgaaggacg gcggccgcta
     1321 cctggtggag ttcaagacca tctacatggc caagaagccc gtgcaactgc ccggctacta
     1381 ctacgtggac accaagctgg acatcacctc ccacaacgag gactacacca tcgtggaaca
     1441 gtacgagcgc tccgagggcc gccaccacct gttcctgtac ggcatggacg agctgtacaa
     1501 ggctagcggc agcggcgcca caaacttctc tctgctaaag caagcaggtg atgttgaaga
     1561 aaaccccggg cctactagta tggcgcgtaa ggtcgatctc acctcctgcg atcgcgagcc
     1621 gatccacatc cccggcagca ttcagccgtg cggctgcctg ctagcctgcg acgcgcaggc
     1681 ggtgcggatc acgcgcatta cggaaaatgc cggcgcgttc tttggacgcg aaactccgcg
     1741 ggtcggtgag ctactcgccg attacttcgg cgagaccgaa gcccatgcgc tgcgcaacgc
     1801 actggcgcag tcctccgatc caaagcgacc ggcgctgatc ttcggttggc gcgacggcct
     1861 gaccggccgc accttcgaca tctcactgca tcgccatgac ggtacatcga tcatcgagtt
     1921 cgagcctgcg gcggccgaac aggccgacaa tccgctgcgg ctgacgcggc agatcatcgc
     1981 gcgcaccaaa gaactgaagt cgctcgaaga gatggccgca cgggtgccgc gctatctgca
     2041 ggcgatgctc ggctatcacc gcgtgatgtt gtaccgcttc gcggacgacg gctccgggat
     2101 ggtgatcggc gaggcgaagc gcagcgacct cgagagcttt ctcggtcagc actttccggc
     2161 gtcgctggtc ccgcagcagg cgcggctact gtacttgaag aacgcgatcc gcgtggtctc
     2221 ggattcgcgc ggcatcagca gccggatcgt gcccgagcac gacgcctccg gcgccgcgct
     2281 cgatctgtcg ttcgcgcacc tgcgcagcat ctcgccctgc catctcgaat ttctgcggaa
     2341 catgggcgtc agcgcctcga tgtcgctgtc gatcatcatt gacggcacgc tatggggatt
     2401 gatcatctgt catcattacg agccgcgtgc cgtgccgatg gcgcagcgcg tcgcggccga
     2461 aatgttcgcc gacttcttat cgctgcactt caccgccgcc caccaccaac gcaagcttgg
     2521 ctccggcgag ggcaggggaa gtcttctaac atgcggggac gtggaggaaa atcccggccc
     2581 actcgagatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     2641 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
     2701 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     2761 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
     2821 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     2881 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     2941 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     3001 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     3061 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     3121 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     3181 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca
     3241 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     3301 caagtaatct agaggagcag tcgacgcggc cgcccaacgc gttggatgca tagcttgagt
     3361 attctatagt gtcacctaaa tagcttggcg taatcatggt catagctgtt tcctgtgtga
     3421 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     3481 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     3541 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     3601 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     3661 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     3721 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     3781 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     3841 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     3901 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     3961 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4021 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4081 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     4141 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     4201 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     4261 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     4321 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     4381 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     4441 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     4501 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     4561 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     4621 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     4681 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     4741 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     4801 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     4861 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     4921 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     4981 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     5041 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     5101 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     5161 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     5221 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     5281 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     5341 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     5401 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     5461 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     5521 ccgcgcacat ttccccgaaa agtgccacct gatgcggtgt gaaataccgc acagatgcgt
     5581 aaggagaaaa taccgcatca ggaaattgta agcgttaata ttttgttaaa attcgcgtta
     5641 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     5701 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     5761 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     5821 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     5881 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     5941 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     6001 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc
     6061 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     6121 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
     6181 tttcccagtc acgacgtt
//
