LOCUS       sequence-301646-        7585 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression plasmid for dimeric soluble ACE2 high affinity 
            variant 2.4 (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-sACE2v2.4(732)-8h
FEATURES             Location/Qualifiers
     source          1..7585
                     /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             2875..2898
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(2942..2959)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2948..3172
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3218..3646
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3305..3324)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3515..3536
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3655..3675)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3660..3989
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3840..3975
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3902..3921
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4056..4850
                     /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(4110..4129)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4720..4739
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5024..5145
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5061..5080)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5115..5134
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5194..5210)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5194..5210)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5207..5229)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5218..5234
                     /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(5242..5248,5249..5266,5267..5272))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5287..5308
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5425..5442)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5596..6184)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5676..5695)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6355..7146,7147..7215))
                     /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     6978..6997
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7216..7320)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7395..7414)
                     /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 acaggccaag tactttttgg acaagtttaa ccacgaagcc
      781 gaagacctgt tctatcaaag ttcacttgct tcttggaatt ataacaccaa tattactgaa
      841 gagaatgtcc aaaacatgaa taatgctggg gacaaatggt ctgccttttt aaaggaacag
      901 tccacaactg cccaaatgta tccactacaa gaaattcaga atctcacagt 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 gactcaagga ttctgggaat attccatgct aacggaccca
     1681 ggaaatgttc agaaagcagt ctgccatccc acagcttggg acctggggaa gggcgacttc
     1741 aggattctta tgtgcacaaa ggtgacaatg gacgacttcc tgacagctca tcatgagatg
     1801 gggcatatcc agtatgatat ggcatatgct 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 tgcagaccaa
     2521 agcatcaaag tgaggataag cctaaaatca gctcttggag ataaagcata tgaatggaac
     2581 gacaatgaaa tgtacctgtt ccgatcatct gttgcatatg ctatgaggca gtacttttta
     2641 aaagtaaaaa atcagatgat tctttttggg gaggaggatg tgcgagtggc taatttgaaa
     2701 ccaagaatct cctttaattt ctttgtcact gcacctaaaa atgtgtctga tatcattcct
     2761 agaactgaag ttgaaaaggc catcaggatg tcccggagcc gtatcaatga tgctttccgt
     2821 ctgaatgaca acagcctaga gtttctgggg atacagccaa cacttggatc cggtcaccat
     2881 caccaccacc atcatcattg ataactcgag tctagagggc ccgtttaaac ccgctgatca
     2941 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     3001 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     3061 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     3121 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     3181 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta
     3241 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     3301 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     3361 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     3421 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     3481 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     3541 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     3601 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg
     3661 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3721 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3781 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     3841 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     3901 ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag
     3961 gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg
     4021 gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg
     4081 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     4141 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     4201 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt
     4261 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     4321 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     4381 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     4441 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     4501 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     4561 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg
     4621 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     4681 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     4741 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     4801 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct
     4861 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
     4921 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     4981 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc
     5041 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     5101 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
     5161 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     5221 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     5281 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     5341 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     5401 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     5461 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     5521 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     5581 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     5641 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5701 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5761 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     5821 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5881 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5941 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     6001 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     6061 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     6121 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     6181 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     6241 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     6301 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     6361 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     6421 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     6481 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     6541 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     6601 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     6661 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     6721 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     6781 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6841 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6901 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6961 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     7021 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     7081 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     7141 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     7201 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     7261 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     7321 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta
     7381 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
     7441 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa
     7501 ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc
     7561 gatgtacggg ccagatatac gcgtt
//
