LOCUS       sequence-386798-        5934 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the protein of smURFP-P2A-GFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5934)
  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 5934)
  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-smURFP-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..5934
                     /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..1383
                     /codon_start=1
                     /product="dimeric small ultra-red fluorescent protein 
                     derived from a cyanobacterial phycobiliprotein (Rodriguez 
                     et al., 2016)"
                     /label=smURFP
                     /translation="MAKTSEQRVNIATLLTENKKKIVDKASQDLWRRHPDLIAPGGIAF
                     SQRDRALCLRDYGWFLHLITFCLLAGDKGPIESIGLISIREMYNSLGVPVPAMMESIRC
                     LKEASLSLLDEEDANETAPYFDYIIKAMS"
     CDS             1408..1464
                     /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(1477..1479,1480..2193)
                     /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(1477..1479,1480..2190)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1519..1540)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1780..1799)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2127..2148
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2218..2806
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2271..2291)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2689..2700)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2849..2970
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2860..2879)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2914..2933
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3132..3261
                     /label=AAV2 ITR
     rep_origin      complement(3498..4086)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3578..3597)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4257..5048,5049..5117))
                     /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     4880..4899
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5118..5222)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5248..5703)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5385..5406)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5597..5616
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   5775..5904
                     /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 caccatggct aagacttccg aacagagggt gaacattgct
     1021 acactgctga cagaaaataa gaagaaaatc gtggataagg cttcccagga tctgtggcgg
     1081 agacacccag acctgatcgc accaggagga attgctttct ctcagaggga ccgcgctctg
     1141 tgcctgcgag attacggctg gttcctgcat ctgatcacct tttgtctgct ggccggagat
     1201 aagggcccca tcgagtctat tgggctgatc agtattcgag aaatgtataa ctcactggga
     1261 gtgcccgtcc ctgcaatgat ggagagcatt agatgcctga aagaagccag cctgtccctg
     1321 ctggacgaag aggacgccaa cgagaccgca ccctactttg attacattat taaggctatg
     1381 agccgaccgg ttcacgtggg aagtggggct acaaacttct cactgctgaa acaggccggg
     1441 gatgtggagg aaaacccagg accacacgtg actagtgtga gcaagggcga ggagctgttc
     1501 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     1561 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     1621 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     1681 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     1741 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     1801 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     1861 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     1921 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     1981 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     2041 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     2101 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     2161 atcactctcg gcatggacga gctgtacaag taagaattcg atatcaagct tatcgataat
     2221 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     2281 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     2341 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     2401 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     2461 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     2521 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     2581 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     2641 tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     2701 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     2761 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgtcga
     2821 cccgggcggc cgcttcgagc agacatgata agatacattg atgagtttgg acaaaccaca
     2881 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
     2941 gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt
     3001 caggttcagg gggagatgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt
     3061 aaaatcgata aggatcttcc tagagcatgg ctacgtagat aagtagcatg gcgggttaat
     3121 cattaactac aaggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     3181 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc
     3241 agtgagcgag cgagcgcgca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3301 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3361 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3421 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     3481 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     3541 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     3601 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     3661 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     3721 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     3781 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     3841 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     3901 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     3961 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4021 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4081 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4141 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4201 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4261 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4321 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     4381 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     4441 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     4501 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     4561 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     4621 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     4681 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     4741 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     4801 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     4861 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     4921 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     4981 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     5041 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     5101 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     5161 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5221 cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg ttaaaattcg
     5281 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     5341 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     5401 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     5461 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     5521 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     5581 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     5641 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     5701 cgtcccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
     5761 cgctattacg ccagctgcgc gctcgctcgc tcactgaggc cgcccgggca aagcccgggc
     5821 gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg
     5881 ccaactccat cactaggggt tccttgtagt taatgattaa cccgccatgc tact
//
