LOCUS       sequence-387133-        6464 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the protein of emiRFP670-P2A-GFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6464)
  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 6464)
  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-emiRFP670-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..6464
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        281..557
                     /label=chicken beta-actin promoter
     primer_bind     836..858
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     920..939
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      978..987
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1938..1994
                     /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(2007..2009,2010..2723)
                     /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(2007..2009,2010..2720)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2049..2070)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2310..2329)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2657..2678
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2748..3336
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2801..2821)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3219..3230)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3379..3500
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3390..3409)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3444..3463
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3662..3791
                     /label=AAV2 ITR
     rep_origin      complement(4028..4616)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4108..4127)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4787..5578,5579..5647))
                     /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     5410..5429
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5648..5752)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5778..6233)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5915..5936)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6127..6146
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6305..6434
                     /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 gsgcgcgcca ggcggggcgg
      421 ggcggggsgr ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg
      481 gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc
      541 gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg
      601 ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc
      661 gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt
      721 ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc ggggggagcg
      781 gctcggggct gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc
      841 ggcttctggc gtgtgaccgg cggctctaga gcctctgcta accatgttca tgccttcttc
      901 tttttcctac agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa
      961 gaattggatc cggtaccgcc accatggccg agggcagcgt ggccagacag cccgacctgc
     1021 tgacctgcga acatgaagag atccacctcg ccggctcgat ccagccgcat ggcgcgcttc
     1081 tggtcgtcag cgaacatgat catcgcgtca tccaggccag cgccaacgcc gcggaatttc
     1141 tgaatctcgg aagcgtactc ggcgttccgc tcgccgagat cgacggcgat ctgttgatca
     1201 agatcctgcc gcatctcgat cccaccgccg aaggcatgcc ggtcgcggtg cgctgccgga
     1261 tcggcaatcc ctctacggag tactgcggtc tgatgcatcg gcctccggaa ggcgggctga
     1321 tcatcgaact cgaacgtgcc ggcccgtcga tcgatctgtc aggcacgctg gcgccggcgc
     1381 tggagcggat ccgcacggcg ggttcactgc gcgcgctgtg cgatgacacc gtgctgctgt
     1441 ttcagcagtg caccggctac gaccgggtga tggtgtatcg tttcgatgag caaggccacg
     1501 gcctggtatt ctccgagtgc catgtgcctg ggctcgaatc ctatttcggc aaccgctatc
     1561 cgtcgtcgac tgtcccgcag atggcgcggc agctgtacgt gcggcagcgc gtccgcgtgc
     1621 tggtcgacgt cacctatcag ccggtgccgc tggagccgcg gctgtcgccg ctgaccgggc
     1681 gcgatctcga catgtcgggc tgcttcctgc gctcgatgtc gccgtgccat ctgcagttcc
     1741 tgaaggacat gggcgtgcgc gccaccctgg cggtgtcgct ggtggtcggc ggcaagctgt
     1801 ggggcctggt tgtctgtcac cattatctgc cgcgcttcat ccgtttcgag ctgcgggcga
     1861 tctgcaaacg gctcgccgaa aggatcgcga cgcggatcac cgcgcttgag agccgaccgg
     1921 ttcacgtggg aagtggggct acaaacttct cactgctgaa acaggccggg gatgtggagg
     1981 aaaacccagg accacacgtg actagtgtga gcaagggcga ggagctgttc accggggtgg
     2041 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     2101 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     2161 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     2221 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     2281 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     2341 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     2401 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     2461 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     2521 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     2581 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     2641 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     2701 gcatggacga gctgtacaag taagaattcg atatcaagct tatcgataat caacctctgg
     2761 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
     2821 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
     2881 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca
     2941 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg
     3001 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg
     3061 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
     3121 attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca
     3181 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc
     3241 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc
     3301 agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgtcga cccgggcggc
     3361 cgcttcgagc agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
     3421 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     3481 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg
     3541 gggagatgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt aaaatcgata
     3601 aggatcttcc tagagcatgg ctacgtagat aagtagcatg gcgggttaat cattaactac
     3661 aaggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     3721 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     3781 cgagcgcgca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     3841 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     3901 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     3961 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     4021 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     4081 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     4141 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     4201 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     4261 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4321 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4381 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4441 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     4501 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4561 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4621 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4681 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     4741 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     4801 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     4861 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     4921 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     4981 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     5041 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     5101 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     5161 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     5221 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     5281 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     5341 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     5401 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     5461 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     5521 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     5581 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     5641 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     5701 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5761 cccgaaaagt gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     5821 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     5881 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     5941 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     6001 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     6061 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     6121 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     6181 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc
     6241 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
     6301 ccagctgcgc gctcgctcgc tcactgaggc cgcccgggca aagcccgggc gtcgggcgac
     6361 ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat
     6421 cactaggggt tccttgtagt taatgattaa cccgccatgc tact
//
