LOCUS       sequence-482833-        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, so these amino acids were substituted by A at 
            Cluster 3 and Cluster 4 of ACE2.
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-C(3+4)
FEATURES             Location/Qualifiers
     source          1..7850
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             362..385
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(436..453)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    442..666
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      712..1140
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(799..818)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1009..1030
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1149..1169)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1154..1483
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1334..1469
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1396..1415
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(1604..1623)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2212..2231
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2516..2637
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2553..2572)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2607..2626
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2686..2702)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2686..2702)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2699..2721)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2710..2726
                     /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(2734..2740,2741..2758,2759..2764))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2779..2800
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2917..2934)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3088..3676)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3168..3187)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3847..4638,4639..4707))
                     /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     4470..4489
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4708..4812)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4887..4906)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5078..5457
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5458..5661
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5612..5632
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5706..5725
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5706..5724
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 tcctgctact gaagttgaag ctgccatcgc aatgtccgca agcgctatca atgacgcttt
       61 cgcactgaat gacaacagcc tagagtttct ggggatacag ccaacacttg gacctcctaa
      121 ccagccccct gtttccatat ggctgattgt ttttggagtt gtgatgggag tgatagtggt
      181 tggcattgtc atcctgatct tcactgggat cagagatcgg aagaagaaaa ataaagcaag
      241 aagtggagaa aatccttatg cctccatcga tattagcaaa ggagaaaata atccaggatt
      301 ccaaaacact gatgatgttc agacctcctt tggtaccgag acctcccagg tggcgcccgc
      361 tgactacaaa gacgatgacg acaagtaatc tagacagtca gcttggatcc gcgttaagtt
      421 taaaccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
      481 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
      541 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
      601 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
      661 ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
      721 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
      781 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
      841 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
      901 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
      961 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     1021 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     1081 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     1141 aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     1201 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     1261 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     1321 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     1381 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca
     1441 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg
     1501 tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     1561 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     1621 ggcaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     1681 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     1741 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     1801 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     1861 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     1921 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     1981 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     2041 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
     2101 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     2161 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     2221 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     2281 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     2341 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     2401 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     2461 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     2521 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     2581 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     2641 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     2701 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     2761 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     2821 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     2881 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     2941 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3001 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3061 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3121 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3181 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     3241 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     3301 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     3361 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     3421 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     3481 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     3541 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     3601 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     3661 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     3721 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     3781 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     3841 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     3901 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     3961 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     4021 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     4081 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     4141 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     4201 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     4261 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     4321 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     4381 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     4441 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     4501 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     4561 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     4621 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     4681 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     4741 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     4801 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct
     4861 cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     4921 tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct
     4981 acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt
     5041 gcgctgcttc gcgatgtacg ggccagatat acgcgttgac attgattatt gactagttat
     5101 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
     5161 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
     5221 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
     5281 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
     5341 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
     5401 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
     5461 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
     5521 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
     5581 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
     5641 gaggtctata taagcagagc tctctggcta actagagaac ccactgctta ctggcttatc
     5701 gaaattaata cgactcacta tagggagacc caagctggct agcgtttaaa cggcagtcag
     5761 ctagccatgt caagctcttc ctggctcctt ctcagccttg ttgctgtaac tgctgctcag
     5821 tccaccattg aggaacaggc caagacattt ttggacaagt ttaaccacga agccgaagac
     5881 ctgttctatc aaagttcact tgcttcttgg aattataaca ccaatattac tgaagagaat
     5941 gtccaaaaca tgaataatgc tggggacaaa tggtctgcct ttttaaagga acagtccaca
     6001 cttgcccaaa tgtatccact acaagaaatt cagaatctca cagtcaagct tcagctgcag
     6061 gctcttcagc aaaatgggtc ttcagtgctc tcagaagaca agagcaaacg gttgaacaca
     6121 attctaaata caatgagcac catctacagt actggaaaag tttgtaaccc agataatcca
     6181 caagaatgct tattacttga accaggtttg aatgaaataa tggcaaacag tttagactac
     6241 aatgagaggc tctgggcttg ggaaagctgg agatctgagg tcggcaagca gctgaggcca
     6301 ttatatgaag agtatgtggt cttgaaaaat gagatggcaa gagcaaatca ttatgaggac
     6361 tatggggatt attggagagg agactatgaa gtaaatgggg tagatggcta tgactacagc
     6421 cgcggccagt tgattgaaga tgtggaacat acctttgaag agattaaacc attatatgaa
     6481 catcttcatg cctatgtgag ggcaaagttg atgaatgcct atccttccta tatcagtcca
     6541 attggatgcc tccctgctca tttgcttggt gatatgtggg gtagattttg gacaaatctg
     6601 tactctttga cagttccctt tggacagaaa ccaaacatag atgttactga tgcaatggtg
     6661 gaccaggcct gggatgcaca gagaatattc aaggaggccg agaagttctt tgtatctgtt
     6721 ggtcttccta atatgactca aggattctgg gaaaattcca tgctaacgga cccaggaaat
     6781 gttcagaaag cagtctgcca tcccacagct tgggacctgg ggaagggcga cttcaggatc
     6841 cttatgtgca caaaggtgac aatggacgac ttcctgacag ctcatcatga gatggggcat
     6901 atccagtatg atatggcata tgctgcacaa ccttttctgc taagaaatgg agctaatgaa
     6961 ggattccatg aagctgttgg ggaaatcatg tcactttctg cagccacacc taagcattta
     7021 aaatccattg gtcttctgtc acccgatttt caagaagaca atgaaacaga aataaacttc
     7081 ctgctcaaac aagcactcac gattgttggg actctgccat ttacttacat gttagagaag
     7141 tggaggtgga tggtctttaa aggggaaatt cccaaagacc agtggatgaa aaagtggtgg
     7201 gagatgaagc gagagatagt tggggtggtg gaacctgtgc cccatgatga aacatactgt
     7261 gaccccgcat ctctgttcca tgtttctaat gattactcat tcattcgata ttacacaagg
     7321 accctttacc aattccagtt tcaagaagca ctttgtcaag cagctaaaca tgaaggccct
     7381 ctgcacaaat gtgacatctc aaactctaca gaagctggac agaaactgtt caatatgctg
     7441 aggcttggaa aatcagaacc ctggacccta gcattggaaa atgttgtagg agcaaagaac
     7501 atgaatgtaa ggccactgct caactacttt gagcccttat ttacctggct gaaagaccag
     7561 aacaagaatt cttttgtggg atggagtacc gactggagtc catatgcaga ccaaagcatc
     7621 aaagtgagga taagcctaaa atcagctctt ggagataaag catatgaatg gaacgacaat
     7681 gaaatgtacc tgttccgatc atctgttgca tatgctatga ggcagtactt tttaaaagta
     7741 aaaaatcaga tgattctttt tggggaggag gatgtgcgag tggctaattt ggctccagct
     7801 atctccttca atttcttcgt cactgcacct gctaatgtgt ctgatatcat
//
