LOCUS       sequence-483854-        7975 bp    DNA     circular SYN 19-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 4 located within the collectrin-like site on ACE2 .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7975)
  TITLE     Zaid Abassi Lab Plasmids 2025
REFERENCE   2  (bases 1 to 7975)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 19, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: ACE2-Mutant-C4
FEATURES             Location/Qualifiers
     source          1..7975
                     /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             7937..7960
                     /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 ctggctctag ccatgtcaag
     5401 ctcttcctgg ctcagtcagc tagccatgtc aagctcttcc tggctcagtc agctagccat
     5461 gtcaagctct tcctggctca gtcagctagc catgtcaagc tcttcctggc tccttctcag
     5521 ccttgttgct gtaactgctg ctcagtccac cattgaggaa caggccaaga catttttgga
     5581 caagtttaac cacgaagccg aagacctgtt ctatcaaagt tcacttgctt cttggaatta
     5641 taacaccaat attactgaag agaatgtcca aaacatgaat aatgctgggg acaaatggtc
     5701 tgccttttta aaggaacagt ccacacttgc ccaaatgtat ccactacaag aaattcagaa
     5761 tctcacagtc aagcttcagc tgcaggctct tcagcaaaat gggtcttcag tgctctcaga
     5821 agacaagagc aaacggttga acacaattct aaatacaatg agcaccatct acagtactgg
     5881 aaaagtttgt aacccagata atccacaaga atgcttatta cttgaaccag gtttgaatga
     5941 aataatggca aacagtttag actacaatga gaggctctgg gcttgggaaa gctggagatc
     6001 tgaggtcggc aagcagctga ggccattata tgaagagtat gtggtcttga aaaatgagat
     6061 ggcaagagca aatcattatg aggactatgg ggattattgg agaggagact atgaagtaaa
     6121 tggggtagat ggctatgact acagccgcgg ccagttgatt gaagatgtgg aacatacctt
     6181 tgaagagatt aaaccattat atgaacatct tcatgcctat gtgagggcaa agttgatgaa
     6241 tgcctatcct tcctatatca gtccaattgg atgcctccct gctcatttgc ttggtgatat
     6301 gtggggtaga ttttggacaa atctgtactc tttgacagtt ccctttggac agaaaccaaa
     6361 catagatgtt actgatgcaa tggtggacca ggcctgggat gcacagagaa tattcaagga
     6421 ggccgagaag ttctttgtat ctgttggtct tcctaatatg actcaaggat tctgggaaaa
     6481 ttccatgcta acggacccag gaaatgttca gaaagcagtc tgccatccca cagcttggga
     6541 cctggggaag ggcgacttca ggatccttat gtgcacaaag gtgacaatgg acgacttcct
     6601 gacagctcat catgagatgg ggcatatcca gtatgatatg gcatatgctg cacaaccttt
     6661 tctgctaaga aatggagcta atgaaggatt ccatgaagct gttggggaaa tcatgtcact
     6721 ttctgcagcc acacctaagc atttaaaatc cattggtctt ctgtcacccg attttcaaga
     6781 agacaatgaa acagaaataa acttcctgct caaacaagca ctcacgattg ttgggactct
     6841 gccatttact tacatgttag agaagtggag gtggatggtc tttaaagggg aaattcccaa
     6901 agaccagtgg atgaaaaagt ggtgggagat gaagcgagag atagttgggg tggtggaacc
     6961 tgtgccccat gatgaaacat actgtgaccc cgcatctctg ttccatgttt ctaatgatta
     7021 ctcattcatt cgatattaca caaggaccct ttaccaattc cagtttcaag aagcactttg
     7081 tcaagcagct aaacatgaag gccctctgca caaatgtgac atctcaaact ctacagaagc
     7141 tggacagaaa ctgttcaata tgctgaggct tggaaaatca gaaccctgga ccctagcatt
     7201 ggaaaatgtt gtaggagcaa agaacatgaa tgtaaggcca ctgctcaact actttgagcc
     7261 cttatttacc tggctgaaag accagaacaa gaattctttt gtgggatgga gtaccgactg
     7321 gagtccatat gcagaccaaa gcatcaaagt gaggataagc ctaaaatcag ctcttggaga
     7381 taaagcatat gaatggaacg acaatgaaat gtacctgttc cgatcatctg ttgcatatgc
     7441 tatgaggcag tactttttaa aagtaaaaaa tcagatgatt ctttttgggg aggaggatgt
     7501 gcgagtggct aatttgaaac caagaatctc ctttaatttc tttgtcactg cacctaaaaa
     7561 tgtgtctgat atcattcctg ctactgaagt tgaagctgcc atcgcaatgt ccgcaagcgc
     7621 tatcaatgac gctttcgcac tgaatgacaa cagcctagag tttctgggga tacagccaac
     7681 acttggacct cctaaccagc cccctgtttc catatggctg attgtttttg gagttgtgat
     7741 gggagtgata gtggttggca ttgtcatcct gatcttcact gggatcagag atcggaagaa
     7801 gaaaaataaa gcaagaagtg gagaaaatcc ttatgcctcc atcgatatta gcaaaggaga
     7861 aaataatcca ggattccaaa acactgatga tgttcagacc tcctttggta ccgagacctc
     7921 ccaggtggcg cccgctgact acaaagacga tgacgacaag taatctagac agtca
//
