LOCUS       sequence-325889-        6077 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 6077)
  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 6077)
  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 deltaN2-EGFP
FEATURES             Location/Qualifiers
     source          1..6077
                     /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    1593..1645
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      1653..1662
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1659..1661,1662..1664,1665..2378)
                     /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(1659..1661,1662..1664,1665..2375)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1704..1725)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1965..1984)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2312..2333
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2501..2622
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2538..2557)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2592..2611
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2629..3084)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2766..2787)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2978..2997
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3111..3215
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3217..3574
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3217..3408
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3240..3260)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3425..3560
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3609..4403
                     /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             3609..4403
                     /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(3663..3682)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4273..4292
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4591..4610)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4635..4682
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5011..5599
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5500..5519
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        5774..6077
                     /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 gccaccatga
      241 ttgcgacggt cagagaggtt ctttccgacc gagaattgca cctctcatgg gaggtcggta
      301 agccgcgccc gcctctgaat aggaactatg tctttacagg ctatcgagtt acaaaaaaca
      361 gtaaggtgca gataggggaa tatacttttg agaaaggtga ctatggggac gccgtagtat
      421 acagggggac aactacctac aaactgaatg tgggcgatta tttcgtgttg actagccata
      481 ctgtcatgcc cctctctgcc ccgaccctcg ttccacagga acattatgtt cgcattactg
      541 ggctgtaccc cacgctcaac atatccgatg aatttagctc caacgtggca aactaccaga
      601 aggtgggaat gcaaaagtat agcacgttgc aaggcccccc tggcacaggt aaatcccact
      661 tcgccattgg cttggcgttg tactacccga gtgcccggat cgtctatact gcatgctctc
      721 atgctgcggt agacgcattg tgtgagaaag ccctgaagta tctgcccatt gacaagtgca
      781 gtcggataat cccagctcgg gcgcgagttg aatgcttcga taagtttaag gtaaatagta
      841 cacttgagca gtatgtattt tgtaccgtca acgctctccc cgaaaccact gcggacatag
      901 tggtatttga tgagatatct atggctacca attatgacct ctcagtagtc aacgcacgct
      961 tgcgagcaaa acactatgta tacatcggag acccagcgca attgcctgct cctaggactc
     1021 tcctcaccaa agggactctt gagcctgaat atttcaactc cgtctgcagg ctgatgaaaa
     1081 ccatcggacc cgatatgttt ctcgggacat gtcgaagatg tcctgccgaa attgtagata
     1141 ctgttagcgc cttggtgtac gataataagc tgaaggccca taaagacaag agcgcccagt
     1201 gtttcaagat gttctataag ggagtaatca cacatgacgt tagttctgcc atcaaccgac
     1261 cgcagatagg cgtggtccgc gagttcctga cccgaaatcc agcgtggagg aaggccgtat
     1321 ttatatcacc ttacaatagc caaaatgccg tggcgtcaaa gattttgggg ctcccgactc
     1381 agacggtgga tagctctcaa gggtcagaat acgattacgt tatattcaca cagacgacag
     1441 agacggcaca cagttgcaac gtcaacagat ttaatgtcgc tataacccgc gccaaggttg
     1501 ggattctttg tataatgtct gacagagacc tctacgacaa gctgcagttc acgtcacttg
     1561 agattcctcg caggaacgtt gcgacgcttc aactcgagct caagcttcga attctgcagt
     1621 cgacggtacc gcgggcccgg gatccaccgg tcgccaccat ggtgagcaag ggcgaggagc
     1681 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     1741 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     1801 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
     1861 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     1921 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     1981 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     2041 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     2101 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
     2161 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     2221 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc
     2281 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     2341 ccgggatcac tctcggcatg gacgagctgt acaagtaaag cggccgcgac tctagatcat
     2401 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     2461 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
     2521 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     2581 gcattctagt tgtggtttgt ccaaactcat caatgtatct taaggcgtaa attgtaagcg
     2641 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     2701 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     2761 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     2821 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     2881 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     2941 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     3001 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     3061 ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     3121 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     3181 gataaatgct tcaataatat tgaaaaagga agagtcctga ggcggaaaga accagctgtg
     3241 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3301 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     3361 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     3421 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     3481 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     3541 aggaggcttt tttggaggcc taggcttttg caaagatcga tcaagagaca ggatgaggat
     3601 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     3661 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     3721 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     3781 atgaactgca agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     3841 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     3901 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     3961 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     4021 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     4081 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca
     4141 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     4201 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     4261 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     4321 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     4381 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     4441 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     4501 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     4561 gttcttcgcc caccctaggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg
     4621 aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt
     4681 tcataaacgc ggggttcggt cccagggctg gcactctgtc gataccccac cgagacccca
     4741 ttggggccaa tacgcccgcg tttcttcctt ttccccaccc caccccccaa gttcgggtga
     4801 aggcccaggg ctcgcagcca acgtcggggc ggcaggccct gccatagcct caggttactc
     4861 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     4921 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     4981 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     5041 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     5101 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     5161 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     5221 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     5281 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     5341 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     5401 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     5461 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     5521 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     5581 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     5641 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     5701 taccgccatg cattagttat taatagtaat caattacggg gtcattagtt catagcccat
     5761 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
     5821 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
     5881 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     5941 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
     6001 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
     6061 tcatcgctat taccatg
//
