LOCUS       sequence-350212-        6802 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient mammalian expression of eGFP. Lacks packaging sequence and
            is used as a negative control transfer plasmid for SARS-CoV-2 
            virus-like particles..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6802)
  AUTHORS   Syed AM, Taha TY, Tabata T, Chen IP, Ciling A, Khalid MM, Sreekumar 
            B, Chen PY, Hayashi JM, Soczek KM, Ott M, Doudna JA
  TITLE     Rapid assessment of SARS-CoV-2 evolved variants using virus-like 
            particles.
  JOURNAL   Science. 2021 Nov 4:eabl6184. doi: 10.1126/science.abl6184.
  PUBMED    34735219
REFERENCE   2  (bases 1 to 6802)
  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"; journalName: "Science. 
            2021 Nov 4:eabl6184. doi: 10.1126/science.abl6184."
COMMENT     Sequence Label: GFP-noPS
FEATURES             Location/Qualifiers
     source          1..6802
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        107..486
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        487..690
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     641..661
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     735..754
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        735..753
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      814..823
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(820..822,823..825,826..1542)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYKG"
     primer_bind     complement(865..886)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1126..1145)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1473..1494
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    1608..2196
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1661..1681)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2079..2090)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2265..2282)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2271..2495
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2541..2969
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2628..2647)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2838..2859
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2978..2998)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2983..3312
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3163..3298
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3225..3244
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3379..4173
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3433..3452)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4043..4062
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4347..4468
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4384..4403)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4438..4457
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4517..4533)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4517..4533)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4530..4552)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4541..4557
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4565..4571,4572..4589,4590..4595))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4610..4631
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4748..4765)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4919..5507)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4999..5018)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5678..6469,6470..6538))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6301..6320
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6539..6643)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6718..6737)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt
       61 aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg
      121 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
      181 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      241 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      301 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      361 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      421 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      481 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      541 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      601 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      661 gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc cactgcttac
      721 tggcttatcg aaattaatac gactcactat agggagaccc aagctggcta gcttgttaga
      781 cgaagcttgg gctgcaggtc cgatccaccg gtcgccacca tggtgagcaa gggcgaggag
      841 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
      901 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
      961 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     1021 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     1081 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     1141 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     1201 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     1261 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     1321 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     1381 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     1441 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     1501 gccgggatca ctctcggcat ggacgagctg tacaagggat aaaatcaaga attcgcttat
     1561 gcggccgcct ctgtgtcgac atataaggat ccactcgacg actcgacaat caacctctgg
     1621 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
     1681 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
     1741 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca
     1801 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg
     1861 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg
     1921 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
     1981 attccgtggt gttgtcgggg aagctgacgt cctttccatg gctgctcgcc tgtgttgcca
     2041 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc
     2101 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcatcttcgc cttcgccctc
     2161 agacgagtcg gatctccctt tgggccgcct ccccgcctgg aattaattcg agctcggtac
     2221 cttgattctc gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc
     2281 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     2341 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     2401 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     2461 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     2521 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     2581 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     2641 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     2701 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     2761 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     2821 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     2881 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     2941 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt
     3001 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3061 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3121 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     3181 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     3241 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     3301 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca
     3361 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     3421 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     3481 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     3541 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
     3601 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     3661 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     3721 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     3781 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     3841 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     3901 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     3961 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     4021 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     4081 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     4141 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     4201 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     4261 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     4321 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     4381 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     4441 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accctccacc tctagctaga
     4501 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     4561 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     4621 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4681 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     4741 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     4801 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4861 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4921 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     4981 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     5041 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     5101 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     5161 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     5221 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     5281 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     5341 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     5401 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     5461 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     5521 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     5581 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     5641 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     5701 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     5761 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     5821 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     5881 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     5941 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     6001 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     6061 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     6121 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     6181 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     6241 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     6301 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     6361 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     6421 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     6481 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     6541 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     6601 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     6661 tgccacctga cctccacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac
     6721 aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt
     6781 cgctgagtag tgcgcgagca aa
//
