LOCUS       sequence-324483-        6305 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 6305)
  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 6305)
  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: Nsp14-EGFP
FEATURES             Location/Qualifiers
     source          1..6305
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      535..544
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    2125..2177
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      2185..2194
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2191..2193,2194..2196,2197..2910)
                     /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(2191..2193,2194..2196,2197..2907)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2236..2257)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2497..2516)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2844..2865
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3033..3154
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3070..3089)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3124..3143
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3161..3616)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3298..3319)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3510..3529
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3643..3747
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3749..4106
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3749..3940
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3772..3792)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3957..4092
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4141..4935
                     /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             4141..4935
                     /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(4195..4214)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4805..4824
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5123..5142)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5167..5214
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5543..6131
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6032..6051
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagccacc
      541 atggctgaaa atgtaacggg cttgtttaaa gactgtagta aagtgatcac aggactccac
      601 cccacacaag cacctaccca cctttccgta gatacgaagt tcaaaacgga aggattgtgt
      661 gtggatatac cagggatacc aaaggatatg acgtaccgaa ggctgatttc catgatgggt
      721 tttaagatga attaccaagt taatggctac cccaacatgt tcatcaccag ggaggaggca
      781 attagacacg taagagcctg gataggcttc gatgttgaag gttgccatgc aacccgggaa
      841 gccgtaggca caaaccttcc gttgcagctt ggcttttcca cgggcgtcaa cctcgttgcc
      901 gtaccgactg gctatgttga cacgccgaac aacaccgatt tctctcgcgt aagtgctaag
      961 cctcctccgg gagatcaatt caagcatctt atacctctca tgtacaaggg actgccgtgg
     1021 aatgtagttc gcattaagat cgtgcagatg ctctccgata ctttgaaaaa tctgagcgac
     1081 agggtggtct tcgttctgtg ggcacatggc ttcgaactta cttctatgaa atactttgta
     1141 aaaatcgggc ccgagcggac atgttgtttg tgcgaccgac gagctacttg ttttagcaca
     1201 gcatctgaca cctacgcatg ctggcaccac agtataggct tcgattacgt ctacaatccc
     1261 tttatgatag acgtacaaca atggggcttt acgggtaact tgcagagtaa tcacgatctt
     1321 tactgccaag ttcatgggaa cgcacacgtg gcctcctgcg atgcgataat gacgaggtgc
     1381 ttggccgtgc acgagtgctt tgttaagcgg gtcgattgga ctatagagta tcccataatc
     1441 ggtgacgaac ttaaaattaa tgctgcatgc aggaaagtgc agcacatggt agtaaaagca
     1501 gcgctcctcg cggataagtt tcctgtgctc cacgatatcg gcaaccctaa ggccattaaa
     1561 tgcgtccccc aagctgatgt agagtggaaa ttctatgacg ctcagccatg tagtgacaaa
     1621 gcatacaaga ttgaagaatt gttttattca tatgcgacac actccgataa gttcactgac
     1681 ggagtttgtc tcttttggaa ctgtaatgtt gataggtatc cggcgaatag catagtgtgt
     1741 cgatttgaca cgcgcgttct ctctaatttg aacctccctg gctgcgacgg ggggtctttg
     1801 tacgtaaaca agcatgcatt tcatacccca gcttttgaca aatccgcttt cgtaaatctg
     1861 aaacaacttc ccttcttcta ttattcagat agcccttgcg aatctcacgg taagcaagtg
     1921 gttagtgaca ttgactatgt accacttaaa agtgcaacgt gcattacacg atgcaatttg
     1981 gggggcgcgg tttgccgaca ccatgccaac gaatacagat tgtacctgga tgcatataac
     2041 atgatgataa gtgcagggtt tagtctttgg gtgtataagc agttcgatac gtataatctc
     2101 tggaacacat ttacccgatt gcagctcgag ctcaagcttc gaattctgca gtcgacggta
     2161 ccgcgggccc gggatccacc ggtcgccacc atggtgagca agggcgagga gctgttcacc
     2221 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     2281 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     2341 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     2401 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     2461 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     2521 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     2581 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     2641 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     2701 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     2761 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     2821 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     2881 actctcggca tggacgagct gtacaagtaa agcggccgcg actctagatc ataatcagcc
     2941 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc
     3001 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     3061 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     3121 gttgtggttt gtccaaactc atcaatgtat cttaaggcgt aaattgtaag cgttaatatt
     3181 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     3241 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     3301 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     3361 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     3421 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     3481 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     3541 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     3601 ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     3661 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     3721 cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt
     3781 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3841 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     3901 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     3961 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     4021 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     4081 tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg atcgtttcgc
     4141 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     4201 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     4261 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     4321 caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     4381 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     4441 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     4501 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     4561 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     4621 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac
     4681 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     4741 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     4801 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     4861 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     4921 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     4981 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     5041 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     5101 cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg
     5161 ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttcataaac
     5221 gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc
     5281 aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag
     5341 ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac
     5401 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     5461 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     5521 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     5581 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     5641 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     5701 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     5761 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     5821 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     5881 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     5941 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     6001 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     6061 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     6121 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     6181 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcca
     6241 tgcattagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
     6301 ttccg
//
