LOCUS       sequence-325888-        6527 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 6527)
  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 6527)
  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: Nsp13 K289A-EGFP
FEATURES             Location/Qualifiers
     source          1..6527
                     /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"
     regulatory      231..240
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    2043..2095
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      2103..2112
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2109..2111,2112..2114,2115..2828)
                     /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(2109..2111,2112..2114,2115..2825)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2154..2175)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2415..2434)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2762..2783
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2951..3072
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2988..3007)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3042..3061
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3079..3534)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3216..3237)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3428..3447
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3561..3665
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3667..4024
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3667..3858
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3690..3710)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3875..4010
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4059..4853
                     /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             4059..4853
                     /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(4113..4132)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4723..4742
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5041..5060)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5085..5132
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5461..6049
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5950..5969
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        6224..6527
                     /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 gccaccatgg
      241 ctgttggggc gtgtgtgttg tgcaacagcc aaacctctct gcgatgcgga gcgtgcatac
      301 ggaggccatt tctttgttgt aaatgttgtt atgatcacgt tattagtacg agccataagt
      361 tggttctgag cgtcaacccc tatgtgtgta acgctccggg ttgcgacgta accgatgtga
      421 cacaacttta cctggggggc atgtcatatt actgcaaaag tcacaaaccc ccaatctcat
      481 ttccgttgtg tgcgaatggc caggtctttg gcctttacaa aaatacttgt gttggttcag
      541 acaacgtaac cgattttaac gcgattgcta catgcgattg gacaaatgct ggtgattata
      601 ttttggctaa tacgtgcacg gaaaggctca aattgtttgc agcggagacc ctcaaggcga
      661 ccgaggaaac ctttaaactg agctacggca ttgcgacggt cagagaggtt ctttccgacc
      721 gagaattgca cctctcatgg gaggtcggta agccgcgccc gcctctgaat aggaactatg
      781 tctttacagg ctatcgagtt acaaaaaaca gtaaggtgca gataggggaa tatacttttg
      841 agaaaggtga ctatggggac gccgtagtat acagggggac aactacctac aaactgaatg
      901 tgggcgatta tttcgtgttg actagccata ctgtcatgcc cctctctgcc ccgaccctcg
      961 ttccacagga acattatgtt cgcattactg ggctgtaccc cacgctcaac atatccgatg
     1021 aatttagctc caacgtggca aactaccaga aggtgggaat gcaaaagtat agcacgttgc
     1081 aaggcccccc tggcacaggt gcctcccact tcgccattgg cttggcgttg tactacccga
     1141 gtgcccggat cgtctatact gcatgctctc atgctgcggt agacgcattg tgtgagaaag
     1201 ccctgaagta tctgcccatt gacaagtgca gtcggataat cccagctcgg gcgcgagttg
     1261 aatgcttcga taagtttaag gtaaatagta cacttgagca gtatgtattt tgtaccgtca
     1321 acgctctccc cgaaaccact gcggacatag tggtatttga tgagatatct atggctacca
     1381 attatgacct ctcagtagtc aacgcacgct tgcgagcaaa acactatgta tacatcggag
     1441 acccagcgca attgcctgct cctaggactc tcctcaccaa agggactctt gagcctgaat
     1501 atttcaactc cgtctgcagg ctgatgaaaa ccatcggacc cgatatgttt ctcgggacat
     1561 gtcgaagatg tcctgccgaa attgtagata ctgttagcgc cttggtgtac gataataagc
     1621 tgaaggccca taaagacaag agcgcccagt gtttcaagat gttctataag ggagtaatca
     1681 cacatgacgt tagttctgcc atcaaccgac cgcagatagg cgtggtccgc gagttcctga
     1741 cccgaaatcc agcgtggagg aaggccgtat ttatatcacc ttacaatagc caaaatgccg
     1801 tggcgtcaaa gattttgggg ctcccgactc agacggtgga tagctctcaa gggtcagaat
     1861 acgattacgt tatattcaca cagacgacag agacggcaca cagttgcaac gtcaacagat
     1921 ttaatgtcgc tataacccgc gccaaggttg ggattctttg tataatgtct gacagagacc
     1981 tctacgacaa gctgcagttc acgtcacttg agattcctcg caggaacgtt gcgacgcttc
     2041 aactcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg gatccaccgg
     2101 tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     2161 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     2221 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     2281 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     2341 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     2401 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     2461 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     2521 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     2581 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     2641 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     2701 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
     2761 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     2821 acaagtaaag cggccgcgac tctagatcat aatcagccat accacatttg tagaggtttt
     2881 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     2941 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     3001 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     3061 caatgtatct taaggcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     3121 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     3181 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     3241 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     3301 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     3361 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     3421 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     3481 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg
     3541 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     3601 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     3661 agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc
     3721 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     3781 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     3841 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     3901 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     3961 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     4021 caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     4081 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     4141 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     4201 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat
     4261 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     4321 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     4381 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     4441 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     4501 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     4561 ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc
     4621 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     4681 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     4741 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     4801 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     4861 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     4921 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     4981 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac
     5041 tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca
     5101 gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg
     5161 gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt
     5221 ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc
     5281 ggcaggccct gccatagcct caggttactc atatatactt tagattgatt taaaacttca
     5341 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     5401 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     5461 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     5521 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     5581 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     5641 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     5701 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     5761 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     5821 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     5881 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     5941 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     6001 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     6061 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     6121 gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat taatagtaat
     6181 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
     6241 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
     6301 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     6361 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     6421 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     6481 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatg
//
