LOCUS       sequence-301648-        7585 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression plasmid for dimeric soluble ACE2 high affinity 
            variant 2 (8his tag).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7585)
  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 7585)
  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(732)-8h
FEATURES             Location/Qualifiers
     source          1..7585
                     /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             2495..2518
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(2562..2579)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2568..2792
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2838..3266
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2925..2944)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3135..3156
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3275..3295)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3280..3609
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3460..3595
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3522..3541
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3676..4470
                     /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(3730..3749)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4340..4359
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4644..4765
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4681..4700)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4735..4754
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4814..4830)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4814..4830)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4827..4849)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4838..4854
                     /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(4862..4868,4869..4886,4887..4892))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4907..4928
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5045..5062)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5216..5804)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5296..5315)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5975..6766,6767..6835))
                     /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     6598..6617
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6836..6940)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7015..7034)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7206..7585
                     /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 tccacaactg 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 tgcagaccaa agcatcaaag tgaggataag
     2161 cctaaaatca gctcttggag ataaagcata tgaatggaac gacaatgaaa tgtacctgtt
     2221 ccgatcatct gttgcatatg ctatgaggca gtacttttta aaagtaaaaa atcagatgat
     2281 tctttttggg gaggaggatg tgcgagtggc taatttgaaa ccaagaatct cctttaattt
     2341 ctttgtcact gcacctaaaa atgtgtctga tatcattcct agaactgaag ttgaaaaggc
     2401 catcaggatg tcccggagcc gtatcaatga tgctttccgt ctgaatgaca acagcctaga
     2461 gtttctgggg atacagccaa cacttggatc cggtcaccat caccaccacc atcatcattg
     2521 ataactcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag
     2581 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     2641 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     2701 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     2761 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     2821 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2881 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     2941 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3001 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     3061 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     3121 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     3181 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     3241 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
     3301 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     3361 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3421 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     3481 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     3541 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     3601 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca agagacagga
     3661 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     3721 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     3781 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     3841 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     3901 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     3961 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     4021 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     4081 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     4141 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     4201 gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     4261 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     4321 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     4381 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     4441 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     4501 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     4561 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     4621 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     4681 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4741 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     4801 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     4861 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     4921 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4981 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     5041 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     5101 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5161 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5221 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5281 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5341 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5401 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5461 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5521 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5581 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5641 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5701 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5761 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5821 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5881 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5941 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     6001 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6061 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6121 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6181 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6241 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6301 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6361 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6421 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6481 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6541 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6601 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6661 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6721 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6781 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6841 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6901 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     6961 cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat
     7021 ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc
     7081 tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat
     7141 gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac
     7201 gcgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca
     7261 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
     7321 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
     7381 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
     7441 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
     7501 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
     7561 cgtattagtc atcgctatta ccatg
//
