LOCUS       sequence-375326-        6085 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian cell line expression plasmids for production of EGFP and 
            FusionRed proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6085)
  AUTHORS   Liang GT, Lai C, Yue Z, Zhang H, Li D, Chen Z, Lu X, Tao L, Subach 
            FV, Piatkevich KD
  TITLE     Enhanced small green fluorescent proteins as a multisensing platform
            for biosensor development.
  JOURNAL   Front Bioeng Biotechnol. 2022 Oct 17;10:1039317. doi: 
            10.3389/fbioe.2022.1039317. eCollection 2022.
  PUBMED    36324888
REFERENCE   2  (bases 1 to 6085)
  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: "Front 
            Bioeng Biotechnol."; date: "2022-10-17"; volume: "10:1039317. doi"; 
            pages: " 10.3389/fbioe.2022.1039317. eCollection 2022"
COMMENT     Sequence Label: pAAV-CAG-EGFP-P2A-FusionRed
FEATURES             Location/Qualifiers
     source          1..6085
                     /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             join(985..987,988..990,991..1701)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(985..987,988..990,991..1701)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1030..1051)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1291..1310)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1638..1659
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1726..1782
                     /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             1795..2493
                     /codon_start=1
                     /product="monomeric red fluorescent protein optimized for 
                     use as a fusion tag (Shemiakina et al., 2012)"
                     /label=FusionRed
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMPMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     VVEGGPLPFAFDILATSFMYGSRTFIKHPPGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKVRGVNFPANGPVMQKKTLGWEASTETMYPADGGLEGACDMAL
                     KLVGGGHLICNLETTYRSKKPATNLKMPGVYNVDHRLERIKEADDETYVEQHEVAVARY
                     STGGAGDGGK"
     misc_feature    2518..3106
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2571..2591)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2989..3000)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3149..3270
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3160..3179)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3214..3233
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(3649..4237)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3729..3748)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4408..5199,5200..5268))
                     /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     5031..5050
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5269..5373)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5399..5854)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5536..5557)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5748..5767
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   5926..6055
                     /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 rggsggggsg
      421 gggsggggsg rggggsgggg sggggsgagg 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 caccatggtg agcaagggcg aggagctgtt caccggggtg
     1021 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     1081 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     1141 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     1201 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     1261 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     1321 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     1381 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     1441 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     1501 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     1561 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     1621 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     1681 ggcatggacg agctgtacaa gcgaccggtt cacgtgggaa gtggggctac aaacttctca
     1741 ctgctgaaac aggccgggga tgtggaggaa aacccaggac cacacgtgac tagtatggtg
     1801 agcgagctga ttaaggagaa catgcccatg aagctgtaca tggagggcac cgtgaacaac
     1861 caccacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac ccagaccatg
     1921 agaatcaagg tcgtcgaggg cggccctctc cccttcgcct tcgacatcct ggctaccagc
     1981 ttcatgtacg gcagcagaac cttcatcaag caccctccgg gcatccccga cttctttaag
     2041 cagtccttcc ctgagggctt cacatgggag agagtcacca catacgaaga cgggggcgtg
     2101 ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa cgtcaaggtt
     2161 agaggggtga acttcccagc caacggccct gtgatgcaga agaaaacact cggctgggag
     2221 gcctccaccg agacgatgta ccctgctgac ggaggactgg aaggcgcatg tgacatggct
     2281 ctgaagctcg tgggaggagg acacctgatc tgcaaccttg agacaacata cagatccaag
     2341 aaacccgcta cgaacctcaa gatgcccggc gtctacaacg tggaccacag actggaaaga
     2401 atcaaggagg ccgacgatga gacatacgtc gagcagcacg aggtggctgt ggccagatac
     2461 tctactggtg gcgctggtga tggaggtaaa taagaattcg atatcaagct tatcgataat
     2521 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     2581 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     2641 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     2701 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     2761 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     2821 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     2881 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     2941 tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     3001 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     3061 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgtcga
     3121 cccgggcggc cgcttcgagc agacatgata agatacattg atgagtttgg acaaaccaca
     3181 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
     3241 gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt
     3301 caggttcagg gggagatgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt
     3361 aaaatcgata aggatcttcc tagagcatgg ctacgtagat aagtagcatg gcgggttaat
     3421 cattaactac aaggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     3481 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     3541 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     3601 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     3661 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     3721 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     3781 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     3841 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     3901 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     3961 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     4021 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     4081 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     4141 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     4201 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4261 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     4321 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     4381 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     4441 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     4501 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     4561 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     4621 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     4681 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     4741 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     4801 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     4861 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     4921 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     4981 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     5041 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     5101 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     5161 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     5221 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     5281 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     5341 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa
     5401 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     5461 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     5521 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     5581 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     5641 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     5701 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     5761 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     5821 cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgccattcag gctgcgcaac
     5881 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctgcg cgctcgctcg
     5941 ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca
     6001 gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg ttccttgtag
     6061 ttaatgatta acccgccatg ctact
//
