LOCUS       sequence-292620-        7942 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression plasmid for sACE2-sfGFP high affinity variant 
            4.
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-sACE2v4-sfGFP
FEATURES             Location/Qualifiers
     source          1..7942
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(32..49)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    38..262
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      308..736
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(395..414)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     605..626
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(745..765)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        750..1079
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      930..1065
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     992..1011
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1146..1940
                     /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(1200..1219)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1810..1829
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2114..2235
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2151..2170)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2205..2224
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2284..2300)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2284..2300)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2297..2319)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2308..2324
                     /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(2332..2338,2339..2356,2357..2362))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2377..2398
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2515..2532)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2686..3274)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2766..2785)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3445..4236,4237..4305))
                     /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     4068..4087
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4306..4410)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4485..4504)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4676..5055
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5056..5259
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5210..5230
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5304..5323
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5304..5322
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             7220..7933
                     /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             7220..7930
                     /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(7247..7275)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     7870..7890
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
ORIGIN
        1 tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca
       61 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
      121 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
      181 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
      241 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg
      301 tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
      361 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
      421 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
      481 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
      541 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
      601 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
      661 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
      721 aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
      781 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
      841 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
      901 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
      961 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg
     1021 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     1081 agctcccggg agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt
     1141 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     1201 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     1261 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     1321 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     1381 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     1441 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     1501 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     1561 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     1621 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc
     1681 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     1741 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     1801 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     1861 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     1921 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
     1981 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     2041 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     2101 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     2161 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     2221 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
     2281 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     2341 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     2401 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     2461 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     2521 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     2581 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     2641 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     2701 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     2761 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     2821 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     2881 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     2941 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     3001 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     3061 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     3121 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     3181 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     3241 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     3301 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     3361 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     3421 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     3481 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     3541 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     3601 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     3661 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     3721 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     3781 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     3841 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     3901 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     3961 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     4021 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     4081 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     4141 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     4201 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     4261 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     4321 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     4381 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     4441 cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat
     4501 gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc
     4561 gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg
     4621 cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat
     4681 tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat
     4741 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
     4801 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
     4861 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
     4921 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
     4981 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
     5041 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt
     5101 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac
     5161 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc
     5221 ggtaggcgtg tacggtggga ggtctatata agcagagctc tctggctaac tagagaaccc
     5281 actgcttact ggcttatcga aattaatacg actcactata gggagaccca agctggctag
     5341 cgccaccatg tcaagctctt cctggctcct tctcagcctt gttgctgtaa ctgctgctca
     5401 gtccaccatt gaggaacagg ccaagacatt tttggacaag tttaacgccg aagccgaaga
     5461 cctgttctat caaagttcac ttgcttcttg gaattataac accaatatta ctgaagagaa
     5521 tgtccaaaac atgaataatg ctggggacaa atggtctgcc tttttaaagg aacagtccac
     5581 aactgcccaa atgtatccac tacaagaaat tcagaatctc acagtcaagc ttcagctgca
     5641 ggctcttcag caaaatgggt cttcagtgct ctcagaagac aagagcaaac ggttgaacac
     5701 aattctaaat acaatgagca ccatctacag tactggaaaa gtttgtaacc cagataatcc
     5761 acaagaatgc ttattacttg aaccaggttt gaatgaaata atggcaaaca gtttagacta
     5821 caatgagagg ctctgggctt gggaaagctg gagatctgag gtcggcaagc agctgaggcc
     5881 attatatgaa gagtatgtgg tcttgaaaaa tgagatggca agagcaaatc attatgagga
     5941 ctatggggat tattggagag gagactatga agtaaatggg gtagatggct atgactacag
     6001 ccgcggccag ttgattgaag atgtggaaca tacctttgaa gagattaaac cattatatga
     6061 acatcttcat gcctatgtga gggcaaagtt gatgaatgcc tatccttcct atatcagtcc
     6121 aattggatgc ctccctgctc atttgcttgg tgatatgtgg ggtagatttt ggacaaatct
     6181 gtactctttg acagttccct ttggacagaa accaaacata gatgttactg atgcaatggt
     6241 ggaccaggcc tgggatgcac agagaatatt caaggaggcc gagaagttct ttgtatctgt
     6301 tggtcttcct aatatgactc aaggattctg ggaatattcc atgctaacgg acccaggaaa
     6361 tgttcagaaa gcagtctgcc atcccacagc ttgggacctg gggaagggcg acttcaggat
     6421 tcttatgtgc acaaaggtga caatggacga cttcctgaca gctcatcatg agatggggca
     6481 tatccagtat gatatggcat atcttgcaca accttttctg ctaagaaatg gagctaatga
     6541 aggattccat gaagctgttg gggaaatcat gtcactttct gcagccacac ctaagcattt
     6601 aaaatccatt ggtcttctgt cacccgattt tcaagaagac aatgaaacag aaataaactt
     6661 cctgctcaaa caagcactca cgattgttgg gactctgcca tttacttaca tgttagagaa
     6721 gtggaggtgg atggtcttta aaggggaaat tcccaaagac cagtggatga aaaagtggtg
     6781 ggagatgaag cgagagatag ttggggtggt ggaacctgtg ccccatgatg aaacatactg
     6841 tgaccccgca tctctgttcc atgtttctaa tgattactca ttcattcgat attacacaag
     6901 gaccctttac caattccagt ttcaagaagc actttgtcaa gcagctaaac atgaaggccc
     6961 tctgcacaaa tgtgacatct caaactctac agaagctgga cagaaactgt tcaatatgct
     7021 gaggcttgga aaatcagaac cctggaccct agcattggaa aatgttgtag gagcaaagaa
     7081 catgaatgta aggccactgc tcaactactt tgagccctta tttacctggc tgaaagacca
     7141 gaacaagaat tcttttgtgg gatggagtac cgactggagt ccatatgcag acggatccgg
     7201 tgggtccggt agtggtggtt ccaaaggaga agaacttttc actggagttg tcccaattct
     7261 tgttgaatta gatggtgatg ttaatgggca caaattttct gtccgtggag agggtgaagg
     7321 tgatgctaca aacggaaaac tcacccttaa atttatttgc actactggaa aactacctgt
     7381 tccatggcca acacttgtca ctactctgac ctatggtgtt caatgctttt cccgttatcc
     7441 ggatcacatg aaacggcatg actttttcaa gagtgccatg cccgaaggtt atgtacagga
     7501 acgcactata tctttcaaag atgacgggac ctacaagacg cgtgctgaag tcaagtttga
     7561 aggtgatacc cttgttaatc gtatcgagtt aaaaggtatt gattttaaag aagatggaaa
     7621 cattctcgga cacaaactgg agtacaactt taactcacac aatgtataca tcacggcaga
     7681 caaacaaaag aatggaatca aagctaactt caaaattcgc cacaacgttg aagatggttc
     7741 cgttcaacta gcagaccatt atcaacaaaa tactccaatt ggcgatggcc ctgtcctttt
     7801 accagacaac cattacctgt cgacacaatc tgtcctttcg aaagatccca acgaaaagcg
     7861 tgaccacatg gtccttcttg agtttgtaac tgctgctggg attacacatg gcatggatga
     7921 gctctacaaa tgataactcg ag
//
