LOCUS       sequence-387203-        6468 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the protein of miRFP670-2-P2A-GFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6468)
  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 6468)
  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-miRFP670-2-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..6468
                     /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..1917
                     /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
                     PRAVPMAQRVAAKRFAERLSTHFTAAHHQR"
     CDS             1942..1998
                     /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(2011..2013,2014..2727)
                     /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(2011..2013,2014..2724)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2053..2074)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2314..2333)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2661..2682
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2752..3340
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2805..2825)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3223..3234)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3383..3504
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3394..3413)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3448..3467
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3666..3795
                     /label=AAV2 ITR
     rep_origin      complement(4032..4620)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4112..4131)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4791..5582,5583..5651))
                     /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     5414..5433
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5652..5756)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5782..6237)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5919..5940)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6131..6150
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6309..6438
                     /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 caccatggcg cgtaaggtcg atctcacctc ctgcgatcgc
     1021 gagccgatcc acatccccgg cagcattcag ccgtgcggct gcctgctagc ctgcgacgcg
     1081 caggcggtgc ggatcacgcg cattacggaa aatgccggcg cgttctttgg acgcgaaact
     1141 ccgcgggtcg gtgagctact cgccgattac ttcggcgaga ccgaagccca tgcgctgcgc
     1201 aacgcactgg cgcagtcctc cgatccaaag cgaccggcgc tgatcttcgg ttggcgcgac
     1261 ggcctgaccg gccgcacctt cgacatctca ctgcatcgcc atgacggtac atcgatcatc
     1321 gagttcgagc ctgcggcggc cgaacaggcc gacaatccgc tgcggctgac gcggcagatc
     1381 atcgcgcgca ccaaagaact gaagtcgctc gaagagatgg ccgcacgggt gccgcgctat
     1441 ctgcaggcga tgctcggcta tcaccgcgtg atgttgtacc gcttcgcgga cgacggctcc
     1501 gggatggtga tcggcgaggc gaagcgcagc gacctcgaga gctttctcgg tcagcacttt
     1561 ccggcgtcgc tggtcccgca gcaggcgcgg ctactgtact tgaagaacgc gatccgcgtg
     1621 gtctcggatt cgcgcggcat cagcagccgg atcgtgcccg agcacgacgc ctccggcgcc
     1681 gcgctcgatc tgtcgttcgc gcacctgcgc agcatctcgc cctgccatct cgaatttctg
     1741 cggaacatgg gcgtcagcgc ctcgatgtcg ctgtcgatca tcattgacgg cacgctatgg
     1801 ggattgatca tctgtcatca ttacgagccg cgtgccgtgc cgatggcgca gcgcgtcgcg
     1861 gccaaaaggt tcgccgagcg cttatcgacg cacttcaccg ccgcccacca ccaacgccga
     1921 ccggttcacg tgggaagtgg ggctacaaac ttctcactgc tgaaacaggc cggggatgtg
     1981 gaggaaaacc caggaccaca cgtgactagt gtgagcaagg gcgaggagct gttcaccggg
     2041 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     2101 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
     2161 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
     2221 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     2281 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     2341 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     2401 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc
     2461 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
     2521 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
     2581 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac
     2641 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
     2701 ctcggcatgg acgagctgta caagtaagaa ttcgatatca agcttatcga taatcaacct
     2761 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     2821 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
     2881 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
     2941 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
     3001 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
     3061 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
     3121 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt
     3181 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
     3241 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
     3301 cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg tcgacccggg
     3361 cggccgcttc gagcagacat gataagatac attgatgagt ttggacaaac cacaactaga
     3421 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     3481 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     3541 cagggggaga tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc
     3601 gataaggatc ttcctagagc atggctacgt agataagtag catggcgggt taatcattaa
     3661 ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     3721 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     3781 cgagcgagcg cgcagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     3841 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     3901 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     3961 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     4021 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     4081 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     4141 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     4201 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     4261 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     4321 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     4381 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     4441 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     4501 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     4561 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     4621 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     4681 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     4741 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     4801 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     4861 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     4921 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     4981 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     5041 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     5101 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     5161 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     5221 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     5281 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     5341 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     5401 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     5461 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     5521 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     5581 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     5641 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     5701 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     5761 tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     5821 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     5881 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     5941 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     6001 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     6061 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     6121 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     6181 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc
     6241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     6301 tacgccagct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg
     6361 cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact
     6421 ccatcactag gggttccttg tagttaatga ttaacccgcc atgctact
//
