LOCUS       sequence-292600-        7942 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression plasmid for sACE2-sfGFP high affinity variant 
            3.
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-sACE2v3-sfGFP
FEATURES             Location/Qualifiers
     source          1..7942
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2545..3258
                     /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             2545..3255
                     /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(2572..2600)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3195..3215
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(3299..3316)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3305..3529
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3575..4003
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3662..3681)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3872..3893
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4012..4032)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4017..4346
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4197..4332
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4259..4278
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4413..5207
                     /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(4467..4486)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5077..5096
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5381..5502
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5418..5437)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5472..5491
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5551..5567)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5551..5567)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5564..5586)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5575..5591
                     /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(5599..5605,5606..5623,5624..5629))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5644..5665
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5782..5799)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5953..6541)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6033..6052)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6712..7503,7504..7572))
                     /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     7335..7354
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7573..7677)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7752..7771)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagcgcca ccatgtcaag ctcttcctgg ctccttctca gccttgttgc tgtaactgct
      721 gctcagtcca ccattgagga acaggtcaag tactttttgg acaagtttaa ccacgaagcc
      781 gaagacctgt tctatcaaag ttcacttgct tcttggaatt ataacaccaa tattactgaa
      841 gagaatgtcc aaaacatgaa taatgctggg gacaaatggt ctgccttttt aaaggaacag
      901 tccacacttg cccaaatgta tccactacaa gaaattcaga atctccaagt caagcttcag
      961 ctgcaggctc ttcagcaaaa tgggtcttca gtgctctcag aagacaagag caaacggttg
     1021 aacacaattc taaatacaat gagcaccatc tacagtactg gaaaagtttg taacccagat
     1081 aatccacaag aatgcttatt acttgaacca ggtttgaatg aaataatggc aaacagttta
     1141 gactacaatg agaggctctg ggcttgggaa agctggagat ctgaggtcgg caagcagctg
     1201 aggccattat atgaagagta tgtggtcttg aaaaatgaga tggcaagagc aaatcattat
     1261 gaggactatg gggattattg gagaggagac tatgaagtaa atggggtaga tggctatgac
     1321 tacagccgcg gccagttgat tgaagatgtg gaacatacct ttgaagagat taaaccatta
     1381 tatgaacatc ttcatgccta tgtgagggca aagttgatga atgcctatcc ttcctatatc
     1441 agtccaattg gatgcctccc tgctcatttg cttggtgata tgtggggtag attttggaca
     1501 aatctgtact ctttgacagt tccctttgga cagaaaccaa acatagatgt tactgatgca
     1561 atggtggacc aggcctggga tgcacagaga atattcaagg aggccgagaa gttctttgta
     1621 tctgttggtc ttcctaatat gactccagga ttctgggaaa attccatgct aacggaccca
     1681 ggaaatgttc agaaagcagt ctgccatccc acagcttggg acctggggaa gggcgacttc
     1741 aggattctta tgtgcacaaa ggtgacaatg gacgacttcc tgacagctca tcatgagatg
     1801 gggcatatcc agtatgatat ggcatatctt gcacaacctt ttctgctaag aaatggagct
     1861 aatgaaggat tccatgaagc tgttggggaa atcatgtcac tttctgcagc cacacctaag
     1921 catttaaaat ccattggtct tctgtcaccc gattttcaag aagacaatga aacagaaata
     1981 aacttcctgc tcaaacaagc actcacgatt gttgggactc tgccatttac ttacatgtta
     2041 gagaagtgga ggtggatggt ctttaaaggg gaaattccca aagaccagtg gatgaaaaag
     2101 tggtgggaga tgaagcgaga gatagttggg gtggtggaac ctgtgcccca tgatgaaaca
     2161 tactgtgacc ccgcatctct gttccatgtt tctaatgatt actcattcat tcgatattac
     2221 acaaggaccc tttaccaatt ccagtttcaa gaagcacttt gtcaagcagc taaacatgaa
     2281 ggccctctgc acaaatgtga catctcaaac tctacagaag ctggacagaa actgttcaat
     2341 atgctgaggc ttggaaaatc agaaccctgg accctagcat tggaaaatgt tgtaggagca
     2401 aagaacatga atgtaaggcc actgctcaac tactttgagc ccttatttac ctggctgaaa
     2461 gaccagaaca agaattcttt tgtgggatgg agtaccgact ggagtccata tgcagacgga
     2521 tccggtgggt ccggtagtgg tggttccaaa ggagaagaac ttttcactgg agttgtccca
     2581 attcttgttg aattagatgg tgatgttaat gggcacaaat tttctgtccg tggagagggt
     2641 gaaggtgatg ctacaaacgg aaaactcacc cttaaattta tttgcactac tggaaaacta
     2701 cctgttccat ggccaacact tgtcactact ctgacctatg gtgttcaatg cttttcccgt
     2761 tatccggatc acatgaaacg gcatgacttt ttcaagagtg ccatgcccga aggttatgta
     2821 caggaacgca ctatatcttt caaagatgac gggacctaca agacgcgtgc tgaagtcaag
     2881 tttgaaggtg atacccttgt taatcgtatc gagttaaaag gtattgattt taaagaagat
     2941 ggaaacattc tcggacacaa actggagtac aactttaact cacacaatgt atacatcacg
     3001 gcagacaaac aaaagaatgg aatcaaagct aacttcaaaa ttcgccacaa cgttgaagat
     3061 ggttccgttc aactagcaga ccattatcaa caaaatactc caattggcga tggccctgtc
     3121 cttttaccag acaaccatta cctgtcgaca caatctgtcc tttcgaaaga tcccaacgaa
     3181 aagcgtgacc acatggtcct tcttgagttt gtaactgctg ctgggattac acatggcatg
     3241 gatgagctct acaaatgata actcgagtct agagggcccg tttaaacccg ctgatcagcc
     3301 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     3361 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     3421 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     3481 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
     3541 gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
     3601 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     3661 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     3721 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     3781 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     3841 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     3901 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     3961 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt
     4021 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     4081 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     4141 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     4201 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     4261 tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct
     4321 tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat
     4381 ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     4441 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     4501 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     4561 agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc
     4621 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     4681 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     4741 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     4801 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     4861 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     4921 tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg
     4981 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     5041 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     5101 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     5161 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg
     5221 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc
     5281 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     5341 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     5401 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     5461 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     5521 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     5581 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     5641 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5701 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5761 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     5821 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5881 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5941 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     6001 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     6061 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     6121 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     6181 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     6241 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     6301 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     6361 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     6421 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     6481 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     6541 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     6601 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     6661 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     6721 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     6781 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     6841 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     6901 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     6961 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     7021 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     7081 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     7141 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     7201 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     7261 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     7321 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     7381 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     7441 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     7501 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     7561 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     7621 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     7681 atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg
     7741 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
     7801 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc
     7861 ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat
     7921 gtacgggcca gatatacgcg tt
//
