LOCUS       sequence-298416-        7942 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression plasmid for sACE2-sfGFP high affinity variant 
            2.2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7942)
  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 7942)
  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-sACE2v2.2-sfGFP
FEATURES             Location/Qualifiers
     source          1..7942
                     /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"
     primer_bind     249..268
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        249..267
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2165..2878
                     /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             2165..2875
                     /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(2192..2220)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2815..2835
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(2919..2936)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2925..3149
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3195..3623
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3282..3301)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3492..3513
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3632..3652)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3637..3966
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3817..3952
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3879..3898
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4033..4827
                     /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(4087..4106)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4697..4716
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5001..5122
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5038..5057)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5092..5111
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5171..5187)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5171..5187)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5184..5206)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5195..5211
                     /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(5219..5225,5226..5243,5244..5249))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5264..5285
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5402..5419)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5573..6161)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5653..5672)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6332..7123,7124..7192))
                     /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     6955..6974
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7193..7297)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7372..7391)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7563..7942
                     /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 agctctctgg ctaactagag aacccactgc ttactggctt
      241 atcgaaatta atacgactca ctatagggag acccaagctg gctagcgcca ccatgtcaag
      301 ctcttcctgg ctccttctca gccttgttgc tgtaactgct gctcagtcca ccattgagga
      361 acaggccaag tactttttgg acaagtttaa ccacgaagcc gaagacctgt tctatcaaag
      421 ttcacttgct tcttggaatt ataacaccaa tattactgaa gagaatgtcc aaaacatgaa
      481 taatgctggg gacaaatggt ctgccttttt aaaggaacag tccacacttg cccaaatgta
      541 tccactacaa gaaattcaga atctcacagt caagcttcag ctgcaggctc ttcagcaaaa
      601 tgggtcttca gtgctctcag aagacaagag caaacggttg aacacaattc taaatacaat
      661 gagcaccatc tacagtactg gaaaagtttg taacccagat aatccacaag aatgcttatt
      721 acttgaacca ggtttgaatg aaataatggc aaacagttta gactacaatg agaggctctg
      781 ggcttgggaa agctggagat ctgaggtcgg caagcagctg aggccattat atgaagagta
      841 tgtggtcttg aaaaatgaga tggcaagagc aaatcattat gaggactatg gggattattg
      901 gagaggagac tatgaagtaa atggggtaga tggctatgac tacagccgcg gccagttgat
      961 tgaagatgtg gaacatacct ttgaagagat taaaccatta tatgaacatc ttcatgccta
     1021 tgtgagggca aagttgatga atgcctatcc ttcctatatc agtccaattg gatgcctccc
     1081 tgctcatttg cttggtgata tgtggggtag attttggaca aatctgtact ctttgacagt
     1141 tccctttgga cagaaaccaa acatagatgt tactgatgca atggtggacc aggcctggga
     1201 tgcacagaga atattcaagg aggccgagaa gttctttgta tctgttggtc ttcctaatat
     1261 gactcaagga ttctgggaat attccatgct aacggaccca ggaaatgttc agaaagcagt
     1321 ctgccatccc acagcttggg acctggggaa gggcgacttc aggattctta tgtgcacaaa
     1381 ggtgacaatg gacgacttcc tgacagctca tcatgagatg gggcatatcc agtatgatat
     1441 ggcatatctt gcacaacctt ttctgctaag aaatggagct aatgaaggat tccatgaagc
     1501 tgttggggaa atcatgtcac tttctgcagc cacacctaag catttaaaat ccattggtct
     1561 tctgtcaccc gattttcaag aagacaatga aacagaaata aacttcctgc tcaaacaagc
     1621 actcacgatt gttgggactc tgccatttac ttacatgtta gagaagtgga ggtggatggt
     1681 ctttaaaggg gaaattccca aagaccagtg gatgaaaaag tggtgggaga tgaagcgaga
     1741 gatagttggg gtggtggaac ctgtgcccca tgatgaaaca tactgtgacc ccgcatctct
     1801 gttccatgtt tctaatgatt actcattcat tcgatattac acaaggaccc tttaccaatt
     1861 ccagtttcaa gaagcacttt gtcaagcagc taaacatgaa ggccctctgc acaaatgtga
     1921 catctcaaac tctacagaag ctggacagaa actgttcaat atgctgaggc ttggaaaatc
     1981 agaaccctgg accctagcat tggaaaatgt tgtaggagca aagaacatga atgtaaggcc
     2041 actgctcaac tactttgagc ccttatttac ctggctgaaa gaccagaaca agaattcttt
     2101 tgtgggatgg agtaccgact ggagtccata tgcagacgga tccggtgggt ccggtagtgg
     2161 tggttccaaa ggagaagaac ttttcactgg agttgtccca attcttgttg aattagatgg
     2221 tgatgttaat gggcacaaat tttctgtccg tggagagggt gaaggtgatg ctacaaacgg
     2281 aaaactcacc cttaaattta tttgcactac tggaaaacta cctgttccat ggccaacact
     2341 tgtcactact ctgacctatg gtgttcaatg cttttcccgt tatccggatc acatgaaacg
     2401 gcatgacttt ttcaagagtg ccatgcccga aggttatgta caggaacgca ctatatcttt
     2461 caaagatgac gggacctaca agacgcgtgc tgaagtcaag tttgaaggtg atacccttgt
     2521 taatcgtatc gagttaaaag gtattgattt taaagaagat ggaaacattc tcggacacaa
     2581 actggagtac aactttaact cacacaatgt atacatcacg gcagacaaac aaaagaatgg
     2641 aatcaaagct aacttcaaaa ttcgccacaa cgttgaagat ggttccgttc aactagcaga
     2701 ccattatcaa caaaatactc caattggcga tggccctgtc cttttaccag acaaccatta
     2761 cctgtcgaca caatctgtcc tttcgaaaga tcccaacgaa aagcgtgacc acatggtcct
     2821 tcttgagttt gtaactgctg ctgggattac acatggcatg gatgagctct acaaatgata
     2881 actcgagtct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg
     2941 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     3001 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     3061 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     3121 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     3181 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     3241 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     3301 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     3361 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     3421 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3481 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     3541 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     3601 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     3661 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3721 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3781 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     3841 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     3901 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     3961 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga
     4021 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     4081 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     4141 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     4201 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     4261 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     4321 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     4381 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     4441 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     4501 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     4561 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     4621 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     4681 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     4741 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     4801 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     4861 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     4921 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     4981 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     5041 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     5101 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     5161 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     5221 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     5281 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5341 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     5401 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     5461 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5521 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5581 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5641 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5701 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5761 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5821 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5881 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5941 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6001 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6061 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6121 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6181 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6241 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     6301 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     6361 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     6421 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6481 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     6541 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     6601 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     6661 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     6721 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6781 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     6841 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6901 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     6961 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     7021 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     7081 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     7141 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     7201 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     7261 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     7321 ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg
     7381 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
     7441 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
     7501 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
     7561 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
     7621 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     7681 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     7741 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     7801 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     7861 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     7921 attagtcatc gctattacca tg
//
