LOCUS Exported 7210 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Mammalian expression plasmid for soluble ACE2 (protease domain) high affinity variant 2. ACCESSION . VERSION . KEYWORDS pcDNA3-sACE2v2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7210) 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 7210) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7210 /organism="synthetic DNA construct" /mol_type="other DNA" 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(2332..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(3445..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" 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 ccaagtactt tttggacaag tttaaccacg 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 acggatcctg 7201 ataactcgag //