LOCUS       sequence-387131-        6483 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the protein of mRhubarb720-P2A-GFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6483)
  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 6483)
  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-mRhubarb720-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..6483
                     /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..1932
                     /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
                     GSWHRHDQLIFLELEPPQRDVAEPQAFFRHTNSAIRRLQAAETLESACAAAAQEVRKIT
                     GFDRVMIYRFASDFSGEVIAEDRCAEVESKLGQHYPASDIPAQARRLYTINPVRIIPDI
                     NYRPVPVTPDLNPVTGRPIDLSFAILRSVSPVHLEFMRNIGMHGTMSISILRGERLWGL
                     IVCHHRTPYYVDLDGRQACELVAQVLARAIGVMEE"
     CDS             1957..2013
                     /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(2026..2028,2029..2742)
                     /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(2026..2028,2029..2739)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2068..2089)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2329..2348)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2676..2697
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2767..3355
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2820..2840)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3238..3249)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3398..3519
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3409..3428)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3463..3482
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3681..3810
                     /label=AAV2 ITR
     rep_origin      complement(4047..4635)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4127..4146)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4806..5597,5598..5666))
                     /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     5429..5448
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5667..5771)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5797..6252)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5934..5955)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6146..6165
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6324..6453
                     /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 aggattggat ccggtaccgc caccatggct gaaggatccg tcgccaggca gcctgacctc
     1021 ttgacctgcg acgatgagcc gatccatatc cccggtgcca tccaaccgca tggactgctg
     1081 ctcgccctcg ccgccgacat gacgatcgtt gccggcagcg acaaccttcc cgaactcacc
     1141 ggactggcga tcggcgccct gatcggccgc tctgcggccg atgtcttcga ctcggagacg
     1201 cacaaccgtc tgacgatcgc cttggccgag cccggggcgg ccgtcggagc accgatcact
     1261 gtcggcttca cgatgcgaaa ggacgcaggc ttcatcggct cctggcatcg ccatgatcag
     1321 ctcatcttcc tcgagctcga gcctccccag cgggacgtcg ccgagccgca ggcgttcttc
     1381 cgccacacca acagcgccat ccgccgcctg caggccgccg aaaccttgga gagcgcctgc
     1441 gccgccgcgg cgcaagaggt gcggaagatt accggcttcg atcgggtgat gatctatcgc
     1501 ttcgcctccg acttcagcgg cgaagtgatc gcagaggatc ggtgcgccga ggtcgagtca
     1561 aaactaggcc agcactatcc tgcctcagat attccggcgc aggcccgtcg gctctatacc
     1621 atcaacccgg tacggatcat tcccgatatc aattatcggc cggtgccggt caccccagac
     1681 ctcaatccgg tcaccgggcg gccgattgat cttagcttcg ccatcctgcg cagcgtctcg
     1741 cccgtccatc tggaattcat gcgcaacata ggcatgcacg gcacgatgtc gatctcgatt
     1801 ttgcgcggcg agcgactgtg gggattgatc gtttgccatc accgaacgcc gtactacgtc
     1861 gatctcgatg gccgccaagc ctgcgagcta gtcgcccagg ttctggcccg ggcgatcggc
     1921 gtgatggaag agcgaccggt tcacgtggga agtggggcta caaacttctc actgctgaaa
     1981 caggccgggg atgtggagga aaacccagga ccacacgtga ctagtgtgag caagggcgag
     2041 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
     2101 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
     2161 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc
     2221 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
     2281 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
     2341 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     2401 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
     2461 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc
     2521 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     2581 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc
     2641 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     2701 gccgccggga tcactctcgg catggacgag ctgtacaagt aagaattcga tatcaagctt
     2761 atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     2821 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     2881 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     2941 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     3001 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     3061 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     3121 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     3181 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     3241 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     3301 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga
     3361 taccgtcgac ccgggcggcc gcttcgagca gacatgataa gatacattga tgagtttgga
     3421 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     3481 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat
     3541 tttatgtttc aggttcaggg ggagatgtgg gaggtttttt aaagcaagta aaacctctac
     3601 aaatgtggta aaatcgataa ggatcttcct agagcatggc tacgtagata agtagcatgg
     3661 cgggttaatc attaactaca aggaacccct agtgatggag ttggccactc cctctctgcg
     3721 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
     3781 ggcggcctca gtgagcgagc gagcgcgcag ctgcattaat gaatcggcca acgcgcgggg
     3841 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     3901 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     3961 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4021 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4081 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4141 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4201 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4261 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4321 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4381 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     4441 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     4501 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     4561 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     4621 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     4681 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     4741 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     4801 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     4861 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     4921 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     4981 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5041 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5101 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5161 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5221 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5281 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5341 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     5401 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     5461 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     5521 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     5581 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     5641 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     5701 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     5761 ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt
     5821 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     5881 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     5941 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     6001 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     6061 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     6121 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     6181 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     6241 tacagggcgc gtcccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
     6301 gggcctcttc gctattacgc cagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa
     6361 agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag
     6421 agggagtggc caactccatc actaggggtt ccttgtagtt aatgattaac ccgccatgct
     6481 act
//
