LOCUS       sequence-478768-        4806 bp    DNA     circular SYN 16-JUL-2025
DEFINITION  Expresses P450-mEGFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4806)
  AUTHORS   Zhang H, Lesnov GD, Subach OM, Zhang W, Kuzmicheva TP, Vlaskina AV, 
            Samygina VR, Chen L, Ye X, Nikolaeva AY, Gabdulkhakov A, Papadaki S,
            Qin W, Borshchevskiy V, Perfilov MM, Gavrikov AS, Drobizhev M, 
            Mishin AS, Piatkevich KD, Subach FV
  TITLE     Bright and stable monomeric green fluorescent protein derived from 
            StayGold.
  JOURNAL   Nat Methods. 2024 Apr;21(4):657-665. doi: 
            10.1038/s41592-024-02203-y. Epub 2024 Feb 26.
  PUBMED    38409224
REFERENCE   2  (bases 1 to 4806)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41592-024-02203-y"; journalName: "Nat Methods"; date: 
            "2024-04"; volume: "21"; issue: "4"; pages: "657-665"
COMMENT     Sequence Label: pCytERM-mEGFP-N1
FEATURES             Location/Qualifiers
     source          1..4806
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      747..756
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(753..755,756..758,759..1472)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(798..819)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1059..1078)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1406..1427
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1594..1715
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1631..1650)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1685..1704
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1722..2177)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1859..1880)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2071..2090
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2204..2308
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2310..2667
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2310..2501
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2333..2353)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2518..2653
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2702..3496
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2702..3496
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2756..2775)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3366..3385
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3684..3703)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3728..3775
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4104..4692
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4593..4612
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctatggac cctgtggtgg tgctggggct ctgtctctcc tgtttgcttc
      661 tcctttcact ctggaaacag agctatgggg gagggaagct tcgaattctg cagtcgacgg
      721 taccgcgggc ccgggatcca ccggtcgcca ccatggtgag caagggcgag gagctgttca
      781 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
      841 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
      901 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
      961 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     1021 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     1081 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     1141 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     1201 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
     1261 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     1321 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc aagctgagca
     1381 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     1441 tcactctcgg catggacgag ctgtacaagt aagcggccgc gactctagat cataatcagc
     1501 cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac
     1561 ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt
     1621 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     1681 agttgtggtt tgtccaaact catcaatgta tcttaaggcg taaattgtaa gcgttaatat
     1741 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     1801 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     1861 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     1921 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     1981 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     2041 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     2101 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     2161 gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     2221 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     2281 gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg
     2341 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     2401 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     2461 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     2521 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     2581 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     2641 ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg
     2701 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     2761 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     2821 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     2881 gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     2941 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     3001 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     3061 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     3121 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     3181 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga
     3241 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     3301 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     3361 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     3421 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     3481 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     3541 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     3601 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     3661 gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc
     3721 gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa
     3781 cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc
     3841 caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca
     3901 gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata
     3961 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     4021 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     4081 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     4141 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     4201 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     4261 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     4321 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     4381 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     4441 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     4501 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     4561 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     4621 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     4681 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     4741 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     4801 atgcat
//
