LOCUS       sequence-482819-        7777 bp    DNA     circular SYN 11-AUG-2025
DEFINITION  To test ACE2 cleavage by TMPRSS2. This protease targets R and K 
            amino acids of ACE2 likely at Cluster 4 of ACE2 in the 
            collectrin-like site. The whole C4 sequence was deleted in this 
            mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7777)
  TITLE     Zaid Abassi Lab Plasmids 2025
REFERENCE   2  (bases 1 to 7777)
  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-DeltaC4
FEATURES             Location/Qualifiers
     source          1..7777
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             298..321
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(360..377)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    366..590
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      636..1064
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(723..742)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     933..954
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1073..1093)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1078..1407
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1258..1393
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1320..1339
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(1528..1547)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2136..2155
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2440..2561
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2477..2496)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2531..2550
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2610..2626)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2610..2626)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2623..2645)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2634..2650
                     /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(2658..2664,2665..2682,2683..2688))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2703..2724
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2841..2858)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3012..3600)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3092..3111)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3771..4562,4563..4631))
                     /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     4394..4413
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4632..4736)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4811..4830)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5002..5381
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5382..5585
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5536..5556
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5630..5649
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5630..5648
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ctgaatgaca acagcctaga gtttctgggg atacagccaa cacttggacc tcctaaccag
       61 ccccctgttt ccatatggct gattgttttt ggagttgtga tgggagtgat agtggttggc
      121 attgtcatcc tgatcttcac tgggatcaga gatcggaaga agaaaaataa agcaagaagt
      181 ggagaaaatc cttatgcctc catcgatatt agcaaaggag aaaataatcc aggattccaa
      241 aacactgatg atgttcagac ctcctttggt accgagacct cccaggtggc gcccgctgac
      301 tacaaagacg atgacgacaa gtaagcttgg atccgcgtta agtttaaacc gctgatcagc
      361 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
      421 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
      481 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
      541 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
      601 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
      661 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
      721 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
      781 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
      841 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
      901 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
      961 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     1021 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     1081 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     1141 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     1201 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     1261 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     1321 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     1381 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     1441 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     1501 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaac agacaatcgg
     1561 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     1621 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct
     1681 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     1741 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     1801 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     1861 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     1921 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     1981 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     2041 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     2101 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     2161 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     2221 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     2281 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     2341 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     2401 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     2461 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     2521 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     2581 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     2641 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     2701 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     2761 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     2821 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     2881 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     2941 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     3001 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     3061 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     3121 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     3181 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     3241 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     3301 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     3361 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     3421 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     3481 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     3541 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     3601 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     3661 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     3721 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     3781 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     3841 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     3901 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     3961 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     4021 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     4081 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     4141 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     4201 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     4261 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     4321 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     4381 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     4441 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     4501 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     4561 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     4621 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     4681 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     4741 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
     4801 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     4861 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
     4921 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
     4981 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
     5041 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     5101 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     5161 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     5221 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     5281 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
     5341 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
     5401 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     5461 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
     5521 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     5581 gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc
     5641 actataggga gacccaagct ggctagcgtt taaacgggcc ctctagacca tgtcaagctc
     5701 ttcctggctc cttctcagcc ttgttgctgt aactgctgct cagtccacca ttgaggaaca
     5761 ggccaagaca tttttggaca agtttaacca cgaagccgaa gacctgttct atcaaagttc
     5821 acttgcttct tggaattata acaccaatat tactgaagag aatgtccaaa acatgaataa
     5881 tgctggggac aaatggtctg cctttttaaa ggaacagtcc acacttgccc aaatgtatcc
     5941 actacaagaa attcagaatc tcacagtcaa gcttcagctg caggctcttc agcaaaatgg
     6001 gtcttcagtg ctctcagaag acaagagcaa acggttgaac acaattctaa atacaatgag
     6061 caccatctac agtactggaa aagtttgtaa cccagataat ccacaagaat gcttattact
     6121 tgaaccaggt ttgaatgaaa taatggcaaa cagtttagac tacaatgaga ggctctgggc
     6181 ttgggaaagc tggagatctg aggtcggcaa gcagctgagg ccattatatg aagagtatgt
     6241 ggtcttgaaa aatgagatgg caagagcaaa tcattatgag gactatgggg attattggag
     6301 aggagactat gaagtaaatg gggtagatgg ctatgactac agccgcggcc agttgattga
     6361 agatgtggaa catacctttg aagagattaa accattatat gaacatcttc atgcctatgt
     6421 gagggcaaag ttgatgaatg cctatccttc ctatatcagt ccaattggat gcctccctgc
     6481 tcatttgctt ggtgatatgt ggggtagatt ttggacaaat ctgtactctt tgacagttcc
     6541 ctttggacag aaaccaaaca tagatgttac tgatgcaatg gtggaccagg cctgggatgc
     6601 acagagaata ttcaaggagg ccgagaagtt ctttgtatct gttggtcttc ctaatatgac
     6661 tcaaggattc tgggaaaatt ccatgctaac ggacccagga aatgttcaga aagcagtctg
     6721 ccatcccaca gcttgggacc tggggaaggg cgacttcagg atccttatgt gcacaaaggt
     6781 gacaatggac gacttcctga cagctcatca tgagatgggg catatccagt atgatatggc
     6841 atatgctgca caaccttttc tgctaagaaa tggagctaat gaaggattcc atgaagctgt
     6901 tggggaaatc atgtcacttt ctgcagccac acctaagcat ttaaaatcca ttggtcttct
     6961 gtcacccgat tttcaagaag acaatgaaac agaaataaac ttcctgctca aacaagcact
     7021 cacgattgtt gggactctgc catttactta catgttagag aagtggaggt ggatggtctt
     7081 taaaggggaa attcccaaag accagtggat gaaaaagtgg tgggagatga agcgagagat
     7141 agttggggtg gtggaacctg tgccccatga tgaaacatac tgtgaccccg catctctgtt
     7201 ccatgtttct aatgattact cattcattcg atattacaca aggacccttt accaattcca
     7261 gtttcaagaa gcactttgtc aagcagctaa acatgaaggc cctctgcaca aatgtgacat
     7321 ctcaaactct acagaagctg gacagaaact gttcaatatg ctgaggcttg gaaaatcaga
     7381 accctggacc ctagcattgg aaaatgttgt aggagcaaag aacatgaatg taaggccact
     7441 gctcaactac tttgagccct tatttacctg gctgaaagac cagaacaaga attcttttgt
     7501 gggatggagt accgactgga gtccatatgc agaccaaagc atcaaagtga ggataagcct
     7561 aaaatcagct cttggagata aagcatatga atggaacgac aatgaaatgt acctgttccg
     7621 atcatctgtt gcatatgcta tgaggcagta ctttttaaaa gtaaaaaatc agatgattct
     7681 ttttggggag gaggatgtgc gagtggctaa tttgaaacca agaatctcct ttaatttctt
     7741 tgtcactgca cctaaaaatg tgtctgatat cattcct
//
