LOCUS       sequence-210067-        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-GFP-Tdtomato-iRFP670
FEATURES             Location/Qualifiers
     source          1..6198
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(34..36,37..39,40..750)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(34..36,37..39,40..750)
                     /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(79..100)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(340..359)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     687..708
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             766..822
                     /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             829..2256
                     /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             2272..2325
                     /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             2332..3267
                     /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(3323..3341)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3324..3341)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3359..3375)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3359..3375)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3372..3394)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3383..3399
                     /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(3407..3413,3414..3431,3432..3437))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3452..3473
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3590..3607)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3761..4349)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3841..3860)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4520..5311,5312..5380))
                     /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     5143..5162
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5381..5485)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5563..6018)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5700..5721)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5912..5931
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6144..6166
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6158..6175
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6159..6175
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6182..6198,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6182..6198,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcgaattg ggcccgacgt cggatccacc gccatggtga gcaagggcga ggagctgttc
       61 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
      121 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
      181 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
      241 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
      301 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
      361 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
      421 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
      481 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
      541 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
      601 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
      661 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
      721 atcactctcg gcatggacga gctgtacaag gctagcggca gcggcgccac aaacttctct
      781 ctgctaaagc aagcaggtga tgttgaagaa aaccccgggc ctactagtat ggtgagcaag
      841 ggcgaggagg tcatcaaaga gttcatgcgc ttcaaggtgc gcatggaggg ctccatgaac
      901 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc
      961 gccaagctga aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtccccc
     1021 cagttcatgt acggctccaa ggcgtacgtg aagcaccccg ccgacatccc cgattacaag
     1081 aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggt
     1141 ctggtgaccg tgacccagga ctcctccctg caggacggca cgctgatcta caaggtgaag
     1201 atgcgcggca ccaacttccc ccccgacggc cccgtaatgc agaagaagac catgggctgg
     1261 gaggcctcca ccgagcgcct gtacccccgc gacggcgtgc tgaagggcga gatccaccag
     1321 gccctgaagc tgaaggacgg cggccactac ctggtggagt tcaagaccat ctacatggcc
     1381 aagaagcccg tgcaactgcc cggctactac tacgtggaca ccaagctgga catcacctcc
     1441 cacaacgagg actacaccat cgtggaacag tacgagcgct ccgagggccg ccaccacctg
     1501 ttcctggggc atggcaccgg cagcaccggc agcggcagct ccggcaccgc ctcctccgag
     1561 gacaacaaca tggccgtcat caaagagttc atgcgcttca aggtgcgcat ggagggctcc
     1621 atgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc
     1681 cagaccgcca agctgaaggt gaccaagggc ggccccctgc ccttcgcctg ggacatcctg
     1741 tccccccagt tcatgtacgg ctccaaggcg tacgtgaagc accccgccga catccccgat
     1801 tacaagaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac
     1861 ggcggtctgg tgaccgtgac ccaggactcc tccctgcagg acggcacgct gatctacaag
     1921 gtgaagatgc gcggcaccaa cttccccccc gacggccccg taatgcagaa gaagaccatg
     1981 ggctgggagg cctccaccga gcgcctgtac ccccgcgacg gcgtgctgaa gggcgagatc
     2041 caccaggccc tgaagctgaa ggacggcggc cgctacctgg tggagttcaa gaccatctac
     2101 atggccaaga agcccgtgca actgcccggc tactactacg tggacaccaa gctggacatc
     2161 acctcccaca acgaggacta caccatcgtg gaacagtacg agcgctccga gggccgccac
     2221 cacctgttcc tgtacggcat ggacgagctg tacaagaagc ttggctccgg cgagggcagg
     2281 ggaagtcttc taacatgcgg ggacgtggag gaaaatcccg gcccactcga gatggcgcgt
     2341 aaggtcgatc tcacctcctg cgatcgcgag ccgatccaca tccccggcag cattcagccg
     2401 tgcggctgcc tgctagcctg cgacgcgcag gcggtgcgga tcacgcgcat tacggaaaat
     2461 gccggcgcgt tctttggacg cgaaactccg cgggtcggtg agctactcgc cgattacttc
     2521 ggcgagaccg aagcccatgc gctgcgcaac gcactggcgc agtcctccga tccaaagcga
     2581 ccggcgctga tcttcggttg gcgcgacggc ctgaccggcc gcaccttcga catctcactg
     2641 catcgccatg acggtacatc gatcatcgag ttcgagcctg cggcggccga acaggccgac
     2701 aatccgctgc ggctgacgcg gcagatcatc gcgcgcacca aagaactgaa gtcgctcgaa
     2761 gagatggccg cacgggtgcc gcgctatctg caggcgatgc tcggctatca ccgcgtgatg
     2821 ttgtaccgct tcgcggacga cggctccggg atggtgatcg gcgaggcgaa gcgcagcgac
     2881 ctcgagagct ttctcggtca gcactttccg gcgtcgctgg tcccgcagca ggcgcggcta
     2941 ctgtacttga agaacgcgat ccgcgtggtc tcggattcgc gcggcatcag cagccggatc
     3001 gtgcccgagc acgacgcctc cggcgccgcg ctcgatctgt cgttcgcgca cctgcgcagc
     3061 atctcgccct gccatctcga atttctgcgg aacatgggcg tcagcgcctc gatgtcgctg
     3121 tcgatcatca ttgacggcac gctatgggga ttgatcatct gtcatcatta cgagccgcgt
     3181 gccgtgccga tggcgcagcg cgtcgcggcc gaaatgttcg ccgacttctt atcgctgcac
     3241 ttcaccgccg cccaccacca acgctaatct agaggagcag tcgacgcggc cgcccaacgc
     3301 gttggatgca tagcttgagt attctatagt gtcacctaaa tagcttggcg taatcatggt
     3361 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     3421 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     3481 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     3541 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     3601 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     3661 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     3721 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     3781 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     3841 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     3901 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     3961 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4021 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4081 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4141 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4201 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4261 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4321 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4381 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4441 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4501 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     4561 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     4621 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     4681 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     4741 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     4801 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     4861 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     4921 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     4981 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5041 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5101 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5161 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5221 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5281 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5341 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     5401 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5461 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gatgcggtgt
     5521 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggaaattgta agcgttaata
     5581 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     5641 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     5701 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     5761 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     5821 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     5881 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     5941 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     6001 cgccgctaca gggcgcgtcc attcgccatt caggctgcgc aactgttggg aagggcgatc
     6061 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
     6121 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt
     6181 gtaatacgac tcactata
//
