LOCUS       sequence-387200-        6495 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the protein of mIFP-P2A-GFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6495)
  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 6495)
  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-mIFP-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..6495
                     /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"
     CDS             985..1944
                     /codon_start=1
                     /product="naturally monomeric infrared fluorescent protein 
                     derived from a truncated bacteriophytochrome (Yu et al., 
                     2015)"
                     /label=mIFP
                     /translation="MSVPLTTSAFGHAFLANCEREQIHLAGSIQPHGILLAVKEPDNVV
                     IQASINAAEFLNTNSVVGRPLRDLGGDLPLQILPHLNGPLHLAPMTLRCTVGSPPRRVD
                     CTIHRPSNGGLIVELEPATKTTNIAPALDGAFHRITSSSSLMGLCDETATIIREITGYD
                     RVMVVRFDEEGNGEILSERRRADLEAFLGNRYPASTIPQIARRLYEHNRVRLLVDVNYT
                     PVPLQPRISPLNGRDLDMSLSCLRSMSPIHQKYMQDMGVGATLVCSLMVSGRLWGLIAC
                     HHYEPRFVPFHIRAAGEALAETCAIRIATLESFAQSQSK"
     CDS             1969..2025
                     /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(2038..2040,2041..2754)
                     /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(2038..2040,2041..2751)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2080..2101)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2341..2360)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2688..2709
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2779..3367
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2832..2852)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3250..3261)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3410..3531
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3421..3440)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3475..3494
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3693..3822
                     /label=AAV2 ITR
     rep_origin      complement(4059..4647)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4139..4158)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4818..5609,5610..5678))
                     /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     5441..5460
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5679..5783)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5809..6264)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5946..5967)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6158..6177
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6336..6465
                     /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 ggcgcgcgcc aggcggggcg
      421 gggcggggcg aggggcgggg 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 caccatgtcg gtaccgctga ctacctcagc attcggccac
     1021 gcgtttctgg ctaactgtga acgcgagcag atccacctgg cgggctccat tcagccgcac
     1081 ggtatcctgc tggctgtgaa agagccggac aacgtggtga tccaggcttc tattaacgct
     1141 gcggagttcc tgaacaccaa ctctgttgtt ggccgtccgc tgcgtgacct gggcggcgat
     1201 ctgcctttgc agatcctgcc gcacctgaac ggcccgctgc acctggctcc gatgaccctg
     1261 cgttgtaccg tgggttctcc gccgcgtcgt gtggactgta ccattcatcg tccgtctaac
     1321 ggcggcctga tcgtagaact ggaaccagca accaagacca ctaacattgc gccggctctg
     1381 gacggtgcgt ttcatcgtat cacttcttca tcctccctga tgggcctgtg tgacgaaacc
     1441 gcgactatta tccgtgagat tactggctac gaccgtgtga tggtagtacg tttcgatgaa
     1501 gagggtaatg gcgaaattct gtccgaacgt cgtcgtgcgg acctggaagc gttcctgggt
     1561 aaccgctacc cggcgtctac tattccgcag atcgctcgtc gcctgtacga acataaccgt
     1621 gttcgcctgc tggtagatgt gaactatact ccggttccgc tacagccgcg catcagcccg
     1681 ctgaacggtc gtgatctgga tatgtccctg tcttgcctgc gctctatgtc cccgatccac
     1741 cagaaataca tgcaggacat gggcgttggc gcgaccctgg tttgctctct gatggtgtct
     1801 ggtcgtctgt ggggtctgat cgcttgccac cactacgaac cgcgcttcgt tccgttccac
     1861 attcgcgctg ctggcgaagc gctggcggaa acttgtgcga tccgcatcgc gacgctggag
     1921 agctttgcac agtctcagtc caaacgaccg gttcacgtgg gaagtggggc tacaaacttc
     1981 tcactgctga aacaggccgg ggatgtggag gaaaacccag gaccacacgt gactagtgtg
     2041 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     2101 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     2161 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     2221 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     2281 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     2341 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     2401 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     2461 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     2521 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     2581 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     2641 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     2701 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaagaattc
     2761 gatatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     2821 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     2881 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
     2941 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
     3001 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
     3061 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     3121 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg
     3181 tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc
     3241 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
     3301 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
     3361 tccccgcatc gataccgtcg acccgggcgg ccgcttcgag cagacatgat aagatacatt
     3421 gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt
     3481 tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac
     3541 aattgcattc attttatgtt tcaggttcag ggggagatgt gggaggtttt ttaaagcaag
     3601 taaaacctct acaaatgtgg taaaatcgat aaggatcttc ctagagcatg gctacgtaga
     3661 taagtagcat ggcgggttaa tcattaacta caaggaaccc ctagtgatgg agttggccac
     3721 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     3781 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcatta atgaatcggc
     3841 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     3901 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3961 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4021 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4081 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4141 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4201 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4261 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4321 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttgr gtccaacccg
     4381 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4441 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     4501 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4561 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4621 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4681 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     4741 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4801 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4861 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     4921 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     4981 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5041 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5101 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     5161 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5221 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5281 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5341 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5401 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     5461 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     5521 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     5581 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     5641 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     5701 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     5761 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg
     5821 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     5881 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     5941 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     6001 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     6061 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     6121 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     6181 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     6241 ttaatgcgcc gctacagggc gcgtcccatt cgccattcag gctgcgcaac tgttgggaag
     6301 ggcgatcggt gcgggcctct tcgctattac gccagctgcg cgctcgctcg ctcactgagg
     6361 ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc
     6421 gagcgcgcag agagggagtg gccaactcca tcactagggg ttccttgtag ttaatgatta
     6481 acccgccatg ctact
//
