LOCUS       sequence-482820-        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 0 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(0+4)
FEATURES             Location/Qualifiers
     source          1..7850
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(161..952,953..1021))
                     /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     784..803
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1022..1126)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1201..1220)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1392..1771
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1772..1975
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1926..1946
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2020..2039
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2020..2038
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4526..4549
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(4600..4617)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4606..4830
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4876..5304
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4963..4982)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5173..5194
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5313..5333)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5318..5647
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5498..5633
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5560..5579
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(5768..5787)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6376..6395
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6680..6801
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6717..6736)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6771..6790
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6850..6866)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6850..6866)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6863..6885)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6874..6890
                     /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(6898..6904,6905..6922,6923..6928))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6943..6964
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7081..7098)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7252..7840)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7332..7351)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
       61 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
      121 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
      181 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
      241 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
      301 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
      361 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
      421 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
      481 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
      541 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
      601 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
      661 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
      721 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
      781 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
      841 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
      901 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
      961 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     1021 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     1081 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     1141 aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag
     1201 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga
     1261 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa
     1321 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag
     1381 atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
     1441 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
     1501 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
     1561 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     1621 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     1681 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     1741 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
     1801 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
     1861 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
     1921 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg
     1981 gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc actataggga
     2041 gacccaagct ggctagcgtt taaacggcag tcagctagcc atgtcaagct cttcctggct
     2101 ccttctcagc cttgttgctg taactgctgc tcagtccacc attgaggaac aggccaagac
     2161 atttttggac aagtttaacc acgaagccga agacctgttc tatcaaagtt cacttgcttc
     2221 ttggaattat aacaccaata ttactgaaga gaatgtccaa aacatgaata atgctgggga
     2281 caaatggtct gcctttttaa aggaacagtc cacacttgcc caaatgtatc cactacaaga
     2341 aattcagaat ctcacagtca agcttcagct gcaggctctt cagcaaaatg ggtcttcagt
     2401 gctctcagaa gacaagagca aacggttgaa cacaattcta aatacaatga gcaccatcta
     2461 cagtactgga aaagtttgta acccagataa tccacaagaa tgcttattac ttgaaccagg
     2521 tttgaatgaa ataatggcaa acagtttaga ctacaatgag aggctctggg cttgggaaag
     2581 ctggagatct gaggtcggca agcagctgag gccattatat gaagagtatg tggtcttgaa
     2641 aaatgagatg gcaagagcaa atcattatga ggactatggg gattattgga gaggagacta
     2701 tgaagtaaat ggggtagatg gctatgacta cagccgcggc cagttgattg aagatgtgga
     2761 acataccttt gaagagatta aaccattata tgaacatctt catgcctatg tgagggcaaa
     2821 gttgatgaat gcctatcctt cctatatcag tccaattgga tgcctccctg ctcatttgct
     2881 tggtgatatg tggggtagat tttggacaaa tctgtactct ttgacagttc cctttggaca
     2941 gaaaccaaac atagatgtta ctgatgcaat ggtggaccag gcctgggatg cacagagaat
     3001 attcaaggag gccgagaagt tctttgtatc tgttggtctt cctaatatga ctcaaggatt
     3061 ctgggaaaat tccatgctaa cggacccagg aaatgttcag aaagcagtct gccatcccac
     3121 agcttgggac ctggggaagg gcgacttcag gatccttatg tgcacaaagg tgacaatgga
     3181 cgacttcctg acagctcatc atgagatggg gcatatccag tatgatatgg catatgctgc
     3241 acaacctttt ctgctaagaa atggagctaa tgaaggattc catgaagctg ttggggaaat
     3301 catgtcactt tctgcagcca cacctaagca tttaaaatcc attggtcttc tgtcacccga
     3361 ttttcaagaa gacaatgaaa cagaaataaa cttcctgctc aaacaagcac tcacgattgt
     3421 tgggactctg ccatttactt acatgttaga gaagtggagg tggatggtct ttaaagggga
     3481 aattcccaaa gaccagtgga tgaaaaagtg gtgggagatg aagcgagaga tagttggggt
     3541 ggtggaacct gtgccccatg atgaaacata ctgtgacccc gcatctctgt tccatgtttc
     3601 taatgattac tcattcattc gatattacac aaggaccctt taccaattcc agtttcaaga
     3661 agcactttgt caagcagcta aacatgaagg ccctctgcac aaatgtgaca tctcaaactc
     3721 tacagaagct ggacagaaac tgttcaatat gctgaggctt ggaaaatcag aaccctggac
     3781 cctagcattg gaaaatgttg taggagcaaa gaacatgaat gtaaggccac tgctcaacta
     3841 ctttgagccc ttatttacct ggctgaaaga ccagaacaag aattcttttg tgggatggag
     3901 taccgactgg agtccatatg cagaccaaag catcgcagtg gcgataagcc tagcatcagc
     3961 tcttggagat aaagcatatg aatggaacga caatgaaatg tacctgttcc gatcatctgt
     4021 tgcatatgct atgaggcagt actttttaaa agtaaaaaat cagatgattc tttttgggga
     4081 ggaggatgtg cgagtggcta atttgaaacc aagaatctcc tttaatttct ttgtcactgc
     4141 acctaaaaat gtgtctgata tcattcctgc tactgaagtt gaagctgcca tcgcaatgtc
     4201 cgcaagcgct atcaatgacg ctttcgcact gaatgacaac agcctagagt ttctggggat
     4261 acagccaaca cttggacctc ctaaccagcc ccctgtttcc atatggctga ttgtttttgg
     4321 agttgtgatg ggagtgatag tggttggcat tgtcatcctg atcttcactg ggatcagaga
     4381 tcggaagaag aaaaataaag caagaagtgg agaaaatcct tatgcctcca tcgatattag
     4441 caaaggagaa aataatccag gattccaaaa cactgatgat gttcagacct cctttggtac
     4501 cgagacctcc caggtggcgc ccgctgacta caaagacgat gacgacaagt aatctagaca
     4561 gtcagcttgg atccgcgtta agtttaaacc gctgatcagc ctcgactgtg ccttctagtt
     4621 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     4681 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     4741 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     4801 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     4861 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     4921 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     4981 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     5041 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     5101 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     5161 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     5221 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     5281 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa
     5341 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     5401 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     5461 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     5521 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     5581 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     5641 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg
     5701 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     5761 ggagaggcta ttcggctatg actgggcaac agacaatcgg ctgctctgat gccgccgtgt
     5821 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     5881 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     5941 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     6001 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     6061 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     6121 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     6181 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     6241 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     6301 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     6361 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     6421 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     6481 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     6541 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     6601 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     6661 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     6721 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     6781 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     6841 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     6901 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     6961 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     7021 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     7081 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     7141 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     7201 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     7261 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     7321 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     7381 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     7441 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     7501 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     7561 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     7621 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     7681 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     7741 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     7801 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
//
