LOCUS       sequence-306284-        6736 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression plasmid for RBD of SARS-CoV-2 protein S (spike)
            fused with sfGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6736)
  AUTHORS   Chan KK, Dorosky D, Sharma P, Abbasi SA, Dye JM, Kranz DM, Herbert 
            AS, Procko E
  TITLE     Engineering human ACE2 to optimize binding to the spike protein of 
            SARS coronavirus 2.
  JOURNAL   Science. 2020 Sep 4;369(6508):1261-1265. doi: 
            10.1126/science.abc0870. Epub 2020 Aug 4.
  PUBMED    32753553
REFERENCE   2  (bases 1 to 6736)
  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: 
            "10.1126/science.abc0870"; journalName: "Science"; date: "2020-09-4-
            4"; volume: "369"; issue: "6508"; pages: "1261-1265"
COMMENT     Sequence Label: pcDNA3-SARS-CoV-2-S-RBD-sfGFP
FEATURES             Location/Qualifiers
     source          1..6736
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        131..510
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        511..714
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     665..685
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     759..778
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        759..777
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1469..2182
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             1469..2179
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(1496..1524)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2119..2139
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(2223..2240)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2229..2453
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2499..2927
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2586..2605)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2796..2817
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2936..2956)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2941..3270
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3121..3256
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3183..3202
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3337..4131
                     /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(3391..3410)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4001..4020
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4305..4426
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4342..4361)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4396..4415
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4475..4491)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4475..4491)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4488..4510)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4499..4515
                     /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(4523..4529,4530..4547,4548..4553))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4568..4589
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4706..4723)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4877..5465)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4957..4976)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5636..6427,6428..6496))
                     /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     6259..6278
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6497..6601)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6676..6695)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat
       61 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
      121 tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
      181 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
      241 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
      301 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
      361 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
      421 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
      481 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
      541 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
      601 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
      661 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg
      721 ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag
      781 acccaagctg gctagcgcca ccatgaagac catcatcgcc ctgagctaca tcttctgcct
      841 ggtgttcgcc actaatcttt gtccgttcgg tgaggttttt aacgcgacaa ggttcgctag
      901 tgtatatgct tggaaccgaa agagaatctc caattgcgta gctgattact ccgttctcta
      961 taacagtgcg tccttttcaa cctttaagtg ttacggcgtt tctccaacga agctgaatga
     1021 tctctgtttt acgaacgtgt atgctgactc tttcgttata cggggggacg aagtgagaca
     1081 gatagcacca ggtcagactg ggaagatagc ggattacaac tataagttgc ccgatgattt
     1141 tacggggtgc gtaatcgcat ggaactcaaa caacctcgac tccaaagtag gtggtaatta
     1201 taattacttg tatcgcctgt ttcgaaagag caatttgaag ccttttgagc gggatatttc
     1261 aaccgaaatt taccaagcag gcagtacgcc atgtaacgga gtagagggat ttaattgcta
     1321 ctttcctctt caatcttatg gctttcaacc aacaaacgga gtggggtatc aaccttatag
     1381 agtggtagta ttgtcctttg agctcctcca cgccccggct acagtttgtg ggcccaaaaa
     1441 gggatccggt gggtccggta gtggtggttc caaaggagaa gaacttttca ctggagttgt
     1501 cccaattctt gttgaattag atggtgatgt taatgggcac aaattttctg tccgtggaga
     1561 gggtgaaggt gatgctacaa acggaaaact cacccttaaa tttatttgca ctactggaaa
     1621 actacctgtt ccatggccaa cacttgtcac tactctgacc tatggtgttc aatgcttttc
     1681 ccgttatccg gatcacatga aacggcatga ctttttcaag agtgccatgc ccgaaggtta
     1741 tgtacaggaa cgcactatat ctttcaaaga tgacgggacc tacaagacgc gtgctgaagt
     1801 caagtttgaa ggtgataccc ttgttaatcg tatcgagtta aaaggtattg attttaaaga
     1861 agatggaaac attctcggac acaaactgga gtacaacttt aactcacaca atgtatacat
     1921 cacggcagac aaacaaaaga atggaatcaa agctaacttc aaaattcgcc acaacgttga
     1981 agatggttcc gttcaactag cagaccatta tcaacaaaat actccaattg gcgatggccc
     2041 tgtcctttta ccagacaacc attacctgtc gacacaatct gtcctttcga aagatcccaa
     2101 cgaaaagcgt gaccacatgg tccttcttga gtttgtaact gctgctggga ttacacatgg
     2161 catggatgag ctctacaaat gataactcga gtctagaggg cccgtttaaa cccgctgatc
     2221 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     2281 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     2341 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     2401 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     2461 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     2521 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     2581 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     2641 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     2701 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     2761 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     2821 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     2881 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     2941 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3001 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     3061 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     3121 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     3181 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     3241 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     3301 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     3361 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     3421 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     3481 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     3541 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     3601 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     3661 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     3721 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     3781 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     3841 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     3901 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     3961 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     4021 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     4081 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     4141 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     4201 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4261 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     4321 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4381 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     4441 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     4501 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     4561 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     4621 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4681 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4741 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4801 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4861 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4921 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4981 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5041 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5101 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5161 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5221 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5281 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5341 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5401 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5461 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5521 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     5581 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5641 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5701 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5761 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5821 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     5881 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     5941 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6001 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6061 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6121 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6181 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6241 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6301 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6361 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6421 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6481 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6541 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6601 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
     6661 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
     6721 tgcttgtgtg ttggag
//
