LOCUS       sequence-387191-        6481 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the protein of miRFP680-P2A-GFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6481)
  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 6481)
  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-miRFP680-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..6481
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        281..556
                     /label=chicken beta-actin promoter
     primer_bind     835..857
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     919..938
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      977..986
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             983..1930
                     /codon_start=1
                     /product="phytochrome-based near-infrared fluorescent 
                     protein, originally termed iRFP (Filonov et al., 2011)"
                     /label=iRFP713
                     /note="name was changed to iRFP713 (Shcherbakova and 
                     Verkhusha, 2013)"
                     /translation="MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGS
                     DNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFI
                     GSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKIT
                     GFDRVMIYRFASDFSGEVIAEDRCAEVESKLGLHYPASTVPAQARRLYTINPVRIIPDI
                     NYRPVPVTPDLNPVTGRPIDLSFAILRSVSPCHLEFMRNIGMHGTMSISILRGERLWGL
                     IVCHHRTPYYVDLDGRQACKRVAERLATQIGVMEE"
     CDS             1955..2011
                     /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(2024..2026,2027..2740)
                     /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(2024..2026,2027..2737)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2066..2087)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2327..2346)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2674..2695
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2765..3353
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2818..2838)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3236..3247)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3396..3517
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3407..3426)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3461..3480
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3679..3808
                     /label=AAV2 ITR
     rep_origin      complement(4045..4633)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4125..4144)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4804..5595,5596..5664))
                     /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     5427..5446
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5665..5769)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5795..6250)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5932..5953)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6144..6163
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6322..6451
                     /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 gggggsgggg gggggggggg ggcgcgcgcc aggcggggcg
      421 gggcggggcg agggggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg
      481 cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg
      541 aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc
      601 cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg
      661 ggacggccct tctcctccgg gctgtaatta gcgcttggtt taatgacggc ttgtttcttt
      721 tctgtggctg cgtgaaagcc ttgaggggct ccgggagggc cctttgtgcg gggggagcgg
      781 ctcggggctg tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg
      841 gcttctggcg tgtgaccggc ggctctagag cctctgctaa ccatgttcat gccttcttct
      901 ttttcctaca gctcctgggc aacgtgctgg ttattgtgct gtctcatcat tttggcaaag
      961 aattggatcc ggtaccgcca ccatggcgga aggctccgtc gccaggcagc ctgacctctt
     1021 gacctgcgac gatgagccga tccatatccc cggtgccatc caaccgcatg gactgctgct
     1081 cgccctcgcc gccgacatga cgatcgttgc cggcagcgac aaccttcccg aactcaccgg
     1141 actggcgatc ggcgccctga tcggccgctc tgcggccgat gtcttcgact cggagacgca
     1201 caaccgtctg acgatcgcct tggccgagcc cggggcggcc gtcggagcac cgatcactgt
     1261 cggcttcacg atgcgaaagg acgcaggctt catcggctcc tggcatcgcc atgatcagct
     1321 catcttcctc gagctcgagc ctccccagcg ggacgtcgcc gagccgcagg cgttcttccg
     1381 ccgcaccaac agcgccatcc gccgccttca ggccgccgaa accttggaaa gcgcctgcgc
     1441 cgccgcggcg caagaggtgc ggaagattac cggcttcgat cgggtgatga tctatcgctt
     1501 cgcctccgac ttcagcggcg aagtgatcgc agaggatcgg tgcgccgagg tcgagtcaaa
     1561 actaggcctg cactatcctg cctcaaccgt gccggcgcag gcccgtcggc tctataccat
     1621 caacccggta cggatcattc ccgatatcaa ttatcggccg gtgccggtca ccccagacct
     1681 caatccggtc accgggcggc cgattgatct tagcttcgcc atcctgcgca gcgtctcgcc
     1741 ctgccatctg gagttcatgc gcaacatagg catgcacggc acgatgtcga tctcgatttt
     1801 gcgcggcgag cgactgtggg gattgatcgt ttgccatcac cgaacgccgt actacgtcga
     1861 tctcgatggc cgccaagcct gcaagagggt cgccgagagg ctggccactc agatcggcgt
     1921 gatggaagag cgaccggttc acgtgggaag tggggctaca aacttctcac tgctgaaaca
     1981 ggccggggat gtggaggaaa acccaggacc acacgtgact agtgtgagca agggcgagga
     2041 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     2101 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     2161 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     2221 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     2281 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     2341 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     2401 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     2461 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     2521 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     2581 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     2641 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     2701 cgccgggatc actctcggca tggacgagct gtacaagtaa gaattcgata tcaagcttat
     2761 cgataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     2821 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     2881 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     2941 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     3001 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     3061 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     3121 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct
     3181 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     3241 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     3301 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata
     3361 ccgtcgaccc gggcggccgc ttcgagcaga catgataaga tacattgatg agtttggaca
     3421 aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc
     3481 tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt
     3541 tatgtttcag gttcaggggg agatgtggga ggttttttaa agcaagtaaa acctctacaa
     3601 atgtggtaaa atcgataagg atcttcctag agcatggcta cgtagataag tagcatggcg
     3661 ggttaatcat taactacaag gaacccctag tgatggagtt ggccactccc tctctgcgcg
     3721 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg
     3781 cggcctcagt gagcgagcga gcgcgcagct gcattaatga atcggccaac gcgcggggag
     3841 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     3901 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     3961 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     4021 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4081 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4141 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     4201 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     4261 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     4321 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     4381 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     4441 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     4501 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     4561 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     4621 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     4681 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     4741 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     4801 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     4861 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     4921 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     4981 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     5041 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     5101 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     5161 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     5221 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     5281 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     5341 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     5401 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     5461 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     5521 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     5581 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     5641 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     5701 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     5761 ggttccgcgc acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta
     5821 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     5881 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     5941 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     6001 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     6061 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     6121 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     6181 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     6241 cagggcgcgt cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
     6301 gcctcttcgc tattacgcca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag
     6361 cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag
     6421 ggagtggcca actccatcac taggggttcc ttgtagttaa tgattaaccc gccatgctac
     6481 t
//
