LOCUS       sequence-387167-        6465 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses the protein of emiRFP703-P2A-GFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6465)
  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 6465)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-emiRFP703-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..6465
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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             1939..1995
                     /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(2008..2010,2011..2724)
                     /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(2008..2010,2011..2721)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2050..2071)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2311..2330)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2658..2679
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2749..3337
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2802..2822)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3220..3231)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3380..3501
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3391..3410)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3445..3464
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3663..3792
                     /label=AAV2 ITR
     rep_origin      complement(4029..4617)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4109..4128)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4788..5579,5580..5648))
                     /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     5411..5430
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5649..5753)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5779..6234)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5916..5937)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6128..6147
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6306..6435
                     /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 ttttgtatyt atttatttyt
      361 ymrtkatttt gtgcagcgry gggggcgggg ggggggsggg ggsgsgsgbs rggsggggsg
      421 gggsggggcg 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 aggattggat ccggtaccgc caccatggcc gagggcagcg tggccagaca gcccgacctg
     1021 ctgacctgcg aacatgaaga gatccacctc gccggctcga tccagccgca tggcgcgctt
     1081 ctggtcgtca gcgaacatga tcatcgcgtc atccaggcca gcgccaacgc cgcggaattt
     1141 ctgaatctcg gaagcgtact cggcgttccg ctcgccgaga tcgacggcga tctgttgatc
     1201 aagatcctac cgcatctcga tcccaccgcc gaaggcatgc cggtcgcggt gcgctgccgg
     1261 atcggcaatc cctctacgga gtactgcggt ctgatgcatc ggcctccgga aggcgggctg
     1321 atcatcgaac tcgaacgtgc cggcccgtcg atcgatctgt caggcacgct ggcgccggcg
     1381 ctggagcgga tccgcacggc gggttcactg cgcgcgctgt gcgatgacac cgtgctgctg
     1441 tttcagcagt gcaccggcta cgaccgggtg atggtgtatc gtttcgatga gcaaggccac
     1501 ggcctggtat tctccgagtg ccatgtgcct gggctcgaat cctatttcgg caaccgctat
     1561 ccgtcgtcgc tggtcccgca gatggcgcgg cagctgtacg tgcggcagcg cgtccgcgtg
     1621 ctggtcgacg tcacctatca gccggtgccg ctggagccgc ggctgtcgcc gctgaccggg
     1681 cgcgatctcg acatgtcggg ctgcttcctg cgctcgatgt cgccgatcca tctgcagttc
     1741 ctgaaggaca tgggcgtgcg cgccaccctg gcggtgtcgc tggtggtcgg cggcaagctg
     1801 tggggcctgg ttgtctgtca ccattatctg ccgcgcttca tccgtttcga gctgcgggcg
     1861 atctgcaaac ggctcgccga aaggatcgcg acgcggatca ccgcgcttga gagccgaccg
     1921 gttcacgtgg gaagtggggc tacaaacttc tcactgctga aacaggccgg ggatgtggag
     1981 gaaaacccag gaccacacgt gactagtgtg agcaagggcg aggagctgtt caccggggtg
     2041 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     2101 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     2161 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     2221 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     2281 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     2341 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     2401 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     2461 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     2521 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     2581 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     2641 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     2701 ggcatggacg agctgtacaa gtaagaattc gatatcaagc ttatcgataa tcaacctctg
     2761 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     2821 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     2881 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     2941 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     3001 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     3061 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     3121 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc
     3181 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     3241 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     3301 cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgtcg acccgggcgg
     3361 ccgcttcgag cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
     3421 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     3481 ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag
     3541 ggggagatgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg taaaatcgat
     3601 aaggatcttc ctagagcatg gctacgtaga taagtagcat ggcgggttaa tcattaacta
     3661 caaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     3721 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     3781 gcgagcgcgc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3841 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     3901 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     3961 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     4021 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     4081 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     4141 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     4201 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     4261 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     4321 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4381 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     4441 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     4501 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4561 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4621 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4681 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     4741 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     4801 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     4861 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     4921 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     4981 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     5041 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     5101 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     5161 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     5221 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     5281 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     5341 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     5401 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     5461 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     5521 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     5581 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     5641 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     5701 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     5761 ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     5821 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     5881 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     5941 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     6001 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     6061 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     6121 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     6181 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt
     6241 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     6301 gccagctgcg cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga
     6361 cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca
     6421 tcactagggg ttccttgtag ttaatgatta acccgccatg ctact
//
