LOCUS       sequence-387134-        6498 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the protein of IFP2-P2A-GFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6498)
  AUTHORS   Zhang H, Papadaki S, Sun X, Wang X, Drobizhev M, Yao L, Rehbock M, 
            Koster RW, Wu L, Namikawa K, Piatkevich KD
  TITLE     Quantitative assessment of near-infrared fluorescent proteins.
  JOURNAL   Nat Methods. 2023 Sep 4. doi: 10.1038/s41592-023-01975-z.
  PUBMED    37666982
REFERENCE   2  (bases 1 to 6498)
  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: "Nat 
            Methods. 2023 Sep 4. doi: 10.1038/s41592-023-01975-z."
COMMENT     Sequence Label: pAAV-CAG-IFP2-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..6498
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        281..558
                     /label=chicken beta-actin promoter
     primer_bind     837..859
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     921..940
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      979..988
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             985..1947
                     /codon_start=1
                     /product="bacteriophytochrome-based monomeric infrared 
                     fluorescent protein (Yu et al., 2014)"
                     /label=IFP2.0
                     /translation="MARDPQPFFPPLYLGGPEITTENCEREPIHIPGSIQPHGALLTAD
                     GHSGEVLQVSLNAATFLGQEPTVLRGQTLAALLPDQWPALQTALPPGCQDALQYRATLD
                     WPAAGHLSLTVHRVAELLILEFEPTEAWDSIGPHALRNAMFALESAPNLRALAEVATQT
                     VRELSGFDRVMLYKFAPDATGEVIAEARREGMQAYLGHRFPASTTPAQARALYTRHLLR
                     LTADTRAAAVPLDPVLNPQTNAPTPLGGAVLRATSPMHMQYLRNMGVGSSLSVSVVVGG
                     QLWGLIVCHHQTPYVLPPDLRTTLEYLGRLLSLQVQRKEA"
     CDS             1972..2028
                     /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(2041..2043,2044..2757)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(2041..2043,2044..2754)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2083..2104)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2344..2363)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2691..2712
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2782..3370
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2835..2855)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3253..3264)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3413..3534
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3424..3443)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3478..3497
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3696..3825
                     /label=AAV2 ITR
     rep_origin      complement(4062..4650)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4142..4161)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4821..5612,5613..5681))
                     /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     5444..5463
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5682..5786)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5812..6267)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5949..5970)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6161..6180
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6339..6468
                     /label=AAV2 ITR
ORIGIN
        1 tatctacgta gccatgctct aggaagagta ccattgacgt caataatgac gtatgttccc
       61 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
      121 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
      181 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
      241 tggcagtaca tctacgtatt agtcatcgct attaccatgg tcgaggtgag ccccacgttc
      301 tgcttcactc tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt
      361 taattatttt gtgcagcgat gggggcgggg gggggggggg ggcgsgcgcc rggsggggsg
      421 gggsggggsg rggggcgggg cggggcgagg cggagaggtg cggcggcagc caatcagagc
      481 ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc ctataaaaag
      541 cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc
      601 gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg
      661 cgggacggcc cttctcctcc gggctgtaat tagcgcttgg tttaatgacg gcttgtttct
      721 tttctgtggc tgcgtgaaag ccttgagggg ctccgggagg gccctttgtg cggggggagc
      781 ggctcggggc tgtccgcggg gggacggctg ccttcggggg ggacggggca gggcggggtt
      841 cggcttctgg cgtgtgaccg gcggctctag agcctctgct aaccatgttc atgccttctt
      901 ctttttccta cagctcctgg gcaacgtgct ggttattgtg ctgtctcatc attttggcaa
      961 agaattggat ccggtaccgc caccatggct cgggaccctc aacctttctt ccctcctctg
     1021 tatctcggag gaccagagat cactactgag aactgcgagc gggaacctat acatatacca
     1081 ggttctattc aacctcatgg agcactgctc acagccgatg gacattctgg cgaggtgctg
     1141 caagtttcac tcaacgcagc cactttcctg ggccaggaac caactgttct gcggggacag
     1201 accctggcag ccctgctgcc agaccagtgg cctgctctgc agactgccct gcctccaggc
     1261 tgccaggacg ccctgcagta tcgcgccacc ctggattggc ccgccgccgg acacctgagc
     1321 ctgaccgtgc atagggtggc cgagctgctg attctggaat tcgagcctac tgaggcatgg
     1381 gactctattg gaccccacgc tctgagaaat gcaatgttcg ctctggagag tgctcctaat
     1441 ctccgggcac tggctgaagt cgcaacccaa acagtccggg aactgtcagg tttcgaccgg
     1501 gtgatgctgt acaagtttgc accagacgca acaggagagg ttattgccga agcaaggcgc
     1561 gagggcatgc aggcttacct cgggcatagg tttcccgcat ccaccacccc tgcacaagct
     1621 agggccctct acacaagaca cctgctccgg ctgaccgcag acaccagggc tgcagcagtg
     1681 cccctcgacc ccgtgctgaa tccccagaca aatgctccta cacctctggg cggagctgtc
     1741 ctcagagcta catccccaat gcacatgcag tacctgagga atatgggagt gggctcctcc
     1801 ctgagcgtca gcgtcgtggt cggcggccag ctgtggggac tgattgtctg ccaccatcag
     1861 acaccctacg tgctgccacc cgatctgcgg accaccctgg agtatctggg gaggctcctg
     1921 tccctgcagg tgcagaggaa agaagcccga ccggttcacg tgggaagtgg ggctacaaac
     1981 ttctcactgc tgaaacaggc cggggatgtg gaggaaaacc caggaccaca cgtgactagt
     2041 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     2101 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     2161 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     2221 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     2281 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     2341 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     2401 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     2461 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     2521 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     2581 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     2641 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     2701 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaagaa
     2761 ttcgatatca agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact
     2821 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     2881 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     2941 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     3001 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     3061 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     3121 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca
     3181 tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     3241 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     3301 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     3361 gcctccccgc atcgataccg tcgacccggg cggccgcttc gagcagacat gataagatac
     3421 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     3481 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     3541 aacaattgca ttcattttat gtttcaggtt cagggggaga tgtgggaggt tttttaaagc
     3601 aagtaaaacc tctacaaatg tggtaaaatc gataaggatc ttcctagagc atggctacgt
     3661 agataagtag catggcgggt taatcattaa ctacaaggaa cccctagtga tggagttggc
     3721 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     3781 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgca ttaatgaatc
     3841 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     3901 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     3961 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4021 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4081 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4141 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4201 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     4261 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4321 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4381 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4441 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4501 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     4561 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     4621 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     4681 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     4741 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     4801 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     4861 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     4921 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     4981 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     5041 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     5101 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     5161 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     5221 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     5281 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     5341 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     5401 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     5461 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     5521 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     5581 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     5641 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     5701 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     5761 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa
     5821 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     5881 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     5941 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     6001 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     6061 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     6121 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     6181 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     6241 cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg
     6301 aagggcgatc ggtgcgggcc tcttcgctat tacgccagct gcgcgctcgc tcgctcactg
     6361 aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg
     6421 agcgagcgcg cagagaggga gtggccaact ccatcactag gggttccttg tagttaatga
     6481 ttaacccgcc atgctact
//
