LOCUS       sequence-325897-        7520 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression and localization.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7520)
  AUTHORS   Miserey-Lenkei S, Trajkovic K, D'Ambrosio JM, Patel AJ, Copic A, 
            Mathur P, Schauer K, Goud B, Albanese V, Gautier R, Subra M, Kovacs 
            D, Barelli H, Antonny B
  TITLE     A comprehensive library of fluorescent constructs of SARS-CoV-2 
            proteins and their initial characterisation in different cell types.
  JOURNAL   Biol Cell. 2021 Jul;113(7):311-328. doi: 10.1111/boc.202000158. Epub
            2021 May 10.
  PUBMED    33666950
REFERENCE   2  (bases 1 to 7520)
  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"; doi: 
            "10.1111/boc.202000158"; journalName: "Biol Cell"; date: "2021-07"; 
            volume: "113"; issue: "7"; pages: "311-328"
COMMENT     Sequence Label: Nsp12-EGFP
FEATURES             Location/Qualifiers
     source          1..7520
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    3036..3088
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      3096..3105
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3102..3104,3105..3107,3108..3821)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3102..3104,3105..3107,3108..3818)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3147..3168)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3408..3427)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3755..3776
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3944..4065
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3981..4000)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4035..4054
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4072..4527)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4209..4230)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4421..4440
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4554..4658
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4660..5017
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4660..4851
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4683..4703)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4868..5003
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5052..5846
                     /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             5052..5846
                     /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(5106..5125)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5716..5735
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6034..6053)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6078..6125
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6454..7042
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6943..6962
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        7217..7520
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gccaccatgt
      241 cagcagacgc acaaagtttt ctgaaccggg tatgcggagt cagtgcggcg aggcttacgc
      301 cctgtggtac gggaacatca acggacgttg tttaccgcgc cttcgacata tacaatgata
      361 aagtcgccgg cttcgcaaag ttccttaaaa cgaattgctg taggttccaa gaaaaggacg
      421 aggatgataa tttgatagac tcctacttcg tggtcaaacg acacacgttt tcaaattatc
      481 agcacgagga aacgatatat aatcttctta aagactgccc tgcagtggca aaacacgact
      541 tctttaagtt ccgcattgac ggggatatgg tcccccatat ctcccgacag agattgacga
      601 aatatacaat ggcggatctg gtgtacgccc tgagacactt cgatgagggc aattgtgata
      661 cactcaagga gattctcgtg acctacaatt gttgcgatga tgactatttc aataagaaag
      721 actggtacga cttcgtcgaa aaccctgata tattgagggt gtacgcaaac ctgggtgaac
      781 gagtaagaca ggcacttctg aaaaccgtcc aattctgcga cgctatgaga aacgcgggca
      841 ttgttggtgt gcttacgctg gataaccaag acctcaatgg taattggtac gattttgggg
      901 acttcatcca aaccactcct ggctcaggcg ttcccgtggt agacagctac tattctttgt
      961 tgatgccaat tcttacactc acaagggccc tgaccgctga gtcacacgtc gatacggact
     1021 tgaccaagcc ctacatcaag tgggatcttc tgaagtacga ctttaccgag gaacgattga
     1081 agctttttga tcgctacttt aaatactggg atcagacgta ccacccaaac tgcgtgaatt
     1141 gcctggatga ccgatgtata ctccattgcg cgaactttaa cgttttgttc tctacagtat
     1201 ttcctccgac gagcttcgga ccgcttgtga gaaagatatt cgtcgacggc gtcccatttg
     1261 ttgtcagcac tggttaccac tttcgcgagt tgggggtggt ccacaatcag gatgtcaatt
     1321 tgcattcctc caggctttct tttaaagagt tgcttgttta tgccgctgac cccgcgatgc
     1381 atgcagcctc tgggaatctt ttgctggaca aaaggacgac gtgctttagc gttgctgccc
     1441 ttacaaataa cgtagcattt caaaccgtaa aacccggtaa ttttaataaa gatttttatg
     1501 attttgcagt ctctaagggt tttttcaagg aggggagtag tgtagagctt aaacattttt
     1561 ttttcgctca ggatggtaac gctgcgatca gcgactacga ctactatcga tacaatctcc
     1621 cgacgatgtg tgatatcagg cagttgttgt tcgttgtaga agtggttgat aagtattttg
     1681 actgttatga cggcggctgt atcaatgcca accaggtcat tgtgaataac ctcgataaat
     1741 ctgccgggtt cccctttaac aagtggggga aggcgcgact gtactacgac agcatgtctt
     1801 acgaagatca ggacgcactc tttgcataca caaagaggaa tgtgataccg actataactc
     1861 aaatgaacct taagtatgca atcagcgcga aaaatcgggc taggactgtt gccggagtta
     1921 gcatatgtag taccatgaca aaccgacagt tccaccagaa gttgctgaaa tcaattgctg
     1981 cgacccgggg tgcgactgta gtaatcggga catcaaagtt ctacggcggg tggcataaca
     2041 tgctcaagac ggtctactca gacgttgaga atccgcatct tatgggttgg gattatccaa
     2101 agtgtgacag ggcgatgccc aacatgttgc ggataatggc ctccttggta cttgcacgga
     2161 aacatacaac ctgctgtagt ttgtcacacc gattttaccg actggcaaac gaatgtgctc
     2221 aggttctcag tgaaatggta atgtgtggcg gctccctcta cgttaagccc ggtggcacaa
     2281 gctctggaga cgcaactact gcctatgcaa acagtgtatt caacatctgc caggctgtca
     2341 cggccaatgt taacgcattg ctttccactg acggaaataa gattgcagac aaatacgtga
     2401 gaaatttgca acatagactc tacgaatgcc tctatcgcaa tcgcgatgtt gatacggact
     2461 tcgttaatga gttctatgct tacttgcgaa agcacttctc catgatgatt ctgtcagacg
     2521 acgctgtagt atgctttaat tcaacgtatg cctcccaggg tctggtcgcg tccataaaaa
     2581 attttaaaag cgtcctgtat taccaaaata acgttttcat gagcgaagcg aaatgttgga
     2641 ccgagactga tcttactaag ggccctcatg agttctgctc acaacacact atgttggtca
     2701 agcagggcga cgattacgtg tatcttcctt atcccgatcc tagtaggatt cttggagcag
     2761 gttgcttcgt ggatgatatc gtcaaaacgg acggcaccct catgatagag aggttcgtct
     2821 ctttggctat agacgcgtat ccgcttacta aacatcctaa ccaagaatac gcggatgtat
     2881 tccatcttta tctgcagtat atcagaaagt tgcatgatga gctgaccggt cacatgttgg
     2941 acatgtatag cgtgatgctt accaatgaca atacttcacg gtactgggaa cctgaatttt
     3001 acgaggcgat gtatacgcct cacaccgtcc tgcagctcga gctcaagctt cgaattctgc
     3061 agtcgacggt accgcgggcc cgggatccac cggtcgccac catggtgagc aagggcgagg
     3121 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     3181 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     3241 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     3301 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     3361 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     3421 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     3481 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     3541 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     3601 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     3661 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     3721 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     3781 ccgccgggat cactctcggc atggacgagc tgtacaagta aagcggccgc gactctagat
     3841 cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct
     3901 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc
     3961 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     4021 actgcattct agttgtggtt tgtccaaact catcaatgta tcttaaggcg taaattgtaa
     4081 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     4141 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     4201 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     4261 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     4321 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     4381 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     4441 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     4501 cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa
     4561 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     4621 cctgataaat gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct
     4681 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     4741 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     4801 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     4861 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     4921 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     4981 gtgaggaggc ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag
     5041 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     5101 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     5161 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     5221 tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     5281 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     5341 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     5401 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     5461 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     5521 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga
     5581 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     5641 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     5701 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     5761 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     5821 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     5881 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     5941 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     6001 ggagttcttc gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga
     6061 aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt
     6121 tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc
     6181 ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg
     6241 tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta
     6301 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     6361 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     6421 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     6481 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     6541 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     6601 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     6661 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     6721 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     6781 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     6841 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     6901 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     6961 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     7021 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     7081 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     7141 tattaccgcc atgcattagt tattaatagt aatcaattac ggggtcatta gttcatagcc
     7201 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
     7261 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
     7321 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
     7381 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
     7441 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
     7501 tagtcatcgc tattaccatg
//
