LOCUS       sequence-482825-        7850 bp    DNA     circular SYN 11-AUG-2025
DEFINITION  To test ACE2 cleavage by TMPRSS2. This protease targets R and K 
            amino acids of ACE2; R was substituted by A at Residue 671 of ACE2 
            in the collectrin-like site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7850)
  TITLE     Zaid Abassi Lab Plasmids 2025
REFERENCE   2  (bases 1 to 7850)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 11, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: ACE2-Mutant-R671A
FEATURES             Location/Qualifiers
     source          1..7850
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(36..53)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    42..266
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      312..740
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(399..418)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     609..630
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(749..769)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        754..1083
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      934..1069
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     996..1015
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(1204..1223)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1812..1831
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2116..2237
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2153..2172)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2207..2226
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2286..2302)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2286..2302)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2299..2321)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2310..2326
                     /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(2334..2340,2341..2358,2359..2364))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2379..2400
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2517..2534)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2688..3276)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2768..2787)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3447..4238,4239..4307))
                     /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     4070..4089
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4308..4412)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4487..4506)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4678..5057
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5058..5261
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5212..5232
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5306..5325
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5306..5324
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             7812..7835
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
ORIGIN
        1 gcttggatcc gcgttaagtt taaaccgctg atcagcctcg actgtgcctt ctagttgcca
       61 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
      121 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
      181 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
      241 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
      301 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
      361 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      421 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      481 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      541 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      601 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      661 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      721 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc
      781 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
      841 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
      901 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
      961 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     1021 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     1081 aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga
     1141 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     1201 aggctattcg gctatgactg ggcaacagac aatcggctgc tctgatgccg ccgtgttccg
     1261 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     1321 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     1381 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     1441 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     1501 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     1561 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     1621 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat
     1681 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     1741 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     1801 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     1861 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     1921 ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg
     1981 cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     2041 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     2101 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     2161 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     2221 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     2281 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     2341 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     2401 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     2461 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     2521 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     2581 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     2641 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     2701 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     2761 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     2821 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     2881 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     2941 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     3001 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     3061 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     3121 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     3181 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     3241 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     3301 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     3361 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     3421 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     3481 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     3541 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     3601 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     3661 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     3721 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     3781 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     3841 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     3901 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     3961 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     4021 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     4081 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     4141 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     4201 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     4261 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     4321 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     4381 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     4441 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
     4501 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
     4561 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
     4621 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
     4681 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
     4741 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
     4801 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
     4861 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     4921 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
     4981 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
     5041 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
     5101 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
     5161 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
     5221 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctctggcta actagagaac
     5281 ccactgctta ctggcttatc gaaattaata cgactcacta tagggagacc caagctggct
     5341 agcgtttaaa cggcagtcag ctagccatgt caagctcttc ctggctcctt ctcagccttg
     5401 ttgctgtaac tgctgctcag tccaccattg aggaacaggc caagacattt ttggacaagt
     5461 ttaaccacga agccgaagac ctgttctatc aaagttcact tgcttcttgg aattataaca
     5521 ccaatattac tgaagagaat gtccaaaaca tgaataatgc tggggacaaa tggtctgcct
     5581 ttttaaagga acagtccaca cttgcccaaa tgtatccact acaagaaatt cagaatctca
     5641 cagtcaagct tcagctgcag gctcttcagc aaaatgggtc ttcagtgctc tcagaagaca
     5701 agagcaaacg gttgaacaca attctaaata caatgagcac catctacagt actggaaaag
     5761 tttgtaaccc agataatcca caagaatgct tattacttga accaggtttg aatgaaataa
     5821 tggcaaacag tttagactac aatgagaggc tctgggcttg ggaaagctgg agatctgagg
     5881 tcggcaagca gctgaggcca ttatatgaag agtatgtggt cttgaaaaat gagatggcaa
     5941 gagcaaatca ttatgaggac tatggggatt attggagagg agactatgaa gtaaatgggg
     6001 tagatggcta tgactacagc cgcggccagt tgattgaaga tgtggaacat acctttgaag
     6061 agattaaacc attatatgaa catcttcatg cctatgtgag ggcaaagttg atgaatgcct
     6121 atccttccta tatcagtcca attggatgcc tccctgctca tttgcttggt gatatgtggg
     6181 gtagattttg gacaaatctg tactctttga cagttccctt tggacagaaa ccaaacatag
     6241 atgttactga tgcaatggtg gaccaggcct gggatgcaca gagaatattc aaggaggccg
     6301 agaagttctt tgtatctgtt ggtcttccta atatgactca aggattctgg gaaaattcca
     6361 tgctaacgga cccaggaaat gttcagaaag cagtctgcca tcccacagct tgggacctgg
     6421 ggaagggcga cttcaggatc cttatgtgca caaaggtgac aatggacgac ttcctgacag
     6481 ctcatcatga gatggggcat atccagtatg atatggcata tgctgcacaa ccttttctgc
     6541 taagaaatgg agctaatgaa ggattccatg aagctgttgg ggaaatcatg tcactttctg
     6601 cagccacacc taagcattta aaatccattg gtcttctgtc acccgatttt caagaagaca
     6661 atgaaacaga aataaacttc ctgctcaaac aagcactcac gattgttggg actctgccat
     6721 ttacttacat gttagagaag tggaggtgga tggtctttaa aggggaaatt cccaaagacc
     6781 agtggatgaa aaagtggtgg gagatgaagc gagagatagt tggggtggtg gaacctgtgc
     6841 cccatgatga aacatactgt gaccccgcat ctctgttcca tgtttctaat gattactcat
     6901 tcattcgata ttacacaagg accctttacc aattccagtt tcaagaagca ctttgtcaag
     6961 cagctaaaca tgaaggccct ctgcacaaat gtgacatctc aaactctaca gaagctggac
     7021 agaaactgtt caatatgctg aggcttggaa aatcagaacc ctggacccta gcattggaaa
     7081 atgttgtagg agcaaagaac atgaatgtaa ggccactgct caactacttt gagcccttat
     7141 ttacctggct gaaagaccag aacaagaatt cttttgtggg atggagtacc gactggagtc
     7201 catatgcaga ccaaagcatc aaagtgagga taagcctaaa atcagctctt ggagataaag
     7261 catatgaatg gaacgacaat gaaatgtacc tgttccgatc atctgttgca tatgctatga
     7321 ggcagtactt tttaaaagta aaaaatcaga tgattctttt tggggaggag gatgtggctg
     7381 tggctaattt gaaaccaaga atctccttta atttctttgt cactgcacct aaaaatgtgt
     7441 ctgatatcat tcctagaact gaagttgaaa aggccatcag gatgtcccgg agccgtatca
     7501 atgatgcttt ccgtctgaat gacaacagcc tagagtttct ggggatacag ccaacacttg
     7561 gacctcctaa ccagccccct gtttccatat ggctgattgt ttttggagtt gtgatgggag
     7621 tgatagtggt tggcattgtc atcctgatct tcactgggat cagagatcgg aagaagaaaa
     7681 ataaagcaag aagtggagaa aatccttatg cctccatcga tattagcaaa ggagaaaata
     7741 atccaggatt ccaaaacact gatgatgttc agacctcctt tggtaccgag acctcccagg
     7801 tggcgcccgc tgactacaaa gacgatgacg acaagtaatc tagacagtca
//
