LOCUS       sequence-254301-        7758 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express soma-localized Ace2_D92N_E199V-mRuby3 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7758)
  AUTHORS   Abdelfattah AS, Valenti R, Zheng J, Wong A, Podgorski K, Koyama M, 
            Kim DS, Schreiter ER
  TITLE     A general approach to engineer positive-going eFRET voltage 
            indicators.
  JOURNAL   Nat Commun. 2020 Jul 10;11(1):3444. doi: 10.1038/s41467-020-17322-1.
  PUBMED    32651384
REFERENCE   2  (bases 1 to 7758)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2020-07-10"; volume: "11(1):3444. doi"; pages: " 
            10.1038/s41467-020-17322-1"
COMMENT     Sequence Label: pCAG-Ace2_D92N_E199V-mRuby3-ST
FEATURES             Location/Qualifiers
     source          1..7758
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      8..17
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1790..1807)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1796..2020
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2066..2494
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2153..2172)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2363..2384
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2503..2523)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2508..2837
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2688..2823
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2750..2769
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2848..3447
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2848..2867)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3344..3364
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3607..3728
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3644..3663)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3698..3717
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3777..3793)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3777..3793)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3790..3812)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3801..3817
                     /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(3825..3831,3832..3849,3850..3855))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3870..3891
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4008..4025)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4179..4767)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4259..4278)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4938..5729,5730..5798))
                     /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     5561..5580
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5799..5903)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        5935..6314
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          6590..7606
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     7530..7552
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     7614..7633
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
ORIGIN
        1 gctagccgcc accatggctg acgtggaaac cgagaccggt atgattgcac agtggattgt
       61 ctttgctatt atggctgctg ctgctattgc ttttggagtg gctgtgcact ttcggccttc
      121 agagctgaag agcgcatact atatcaacat tgccatctgc actatcgccg ctaccgctta
      181 ctatgcaatg gccgtgaact accaggacct gacaatgaat ggtgaaaggc aggtggtcta
      241 cgcaagatat attgactggg tgctgaccac accactgctc ctgctcaacc tcatcgtcat
      301 gaccaagatg ggcggagtga tgatttcttg ggtcatcggc gcagacattt tcatgatcgt
      361 gtttggtatt ctgggcgcct tcgaggatga acacaagttc aaatgggtgt actttatcgc
      421 tggatgtgtg atgcaggcag tcctgacata cgggatgtat aacgccactt ggaaagacga
      481 tctgaagaaa agccccgagt accatagctc ctatgtcagt ctgctcgtct tcctgtcaat
      541 cctctgggtg ttttatcctg tcgtgtgggc tttcgggtct ggtagtggcg tgctgtccgt
      601 cgacaatgtg gccattctca tgggaatcct ggatgtgctc gctaagccac tgtttggaat
      661 ggggtgcctc attgcccatg agactatctt caagtggagg tccggactga tcaaggaaaa
      721 tatgcgtatg aaggtggtca tggaaggttc ggtcaacggc caccaattca aatgcacagg
      781 tgaaggagaa ggcaggccgt acgagggagt gcaaaccatg aggatcaaag tcatcgaggg
      841 aggacccctg ccatttgcct ttgacattct tgccacgtcg ttcatgtatg gcagccgtac
      901 ttttatcaag tacccggcag acattcctga tttctttaaa cagtcctttc ctgagggttt
      961 tacttgggaa agagttacga gatacgaaga tggtggagtc gtcaccgtca cgcaggacac
     1021 cagccttgag gatggcgaac tcgtttacaa cgtcaaagtc agaggggtaa actttccctc
     1081 caatggtccc gtgatgcaga agaagaccaa gggttgggag cctaatacag agatgatgta
     1141 tccagcagat ggtggtctga ggggatacac tgatattgca ctgaaagttg atggtggtgg
     1201 ccatctgcat tgcaatttcg taacaactta caggtcaaaa aagaccgtcg ggaacatcaa
     1261 gatgcccggt gtccatgccg ttgatcaccg cctggaaagg atagaggaaa gtgacaatga
     1321 aacgtacgta gtacaacgcg aagtcgcagt tgccaagtac tccaatcttg gtggtggtat
     1381 ggatgaactt tataagaaga gcaggatcac cagcgagggc gagtacatcc ccctggacca
     1441 gatcgatatc aacgtgttct gctacgagaa cgaggtgcaa agtcagccta tcctgaacac
     1501 aaaggaaatg gctccacagt ctaagcctcc cgaagagctt gagatgtcca gtatgccaag
     1561 tcccgtggct cccctccctg ccaggactga aggagtgatt gacatgagga gtatgtcatc
     1621 tattgatagc ttcatctctt gcgcaacaga tttccccgag gctactcgat tctaatgaaa
     1681 gcttgctacc gagctcggat ccactagtcc agtgtggtgg aattctgcag aaatccagca
     1741 cagtggcggc cgctcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg
     1801 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     1861 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     1921 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     1981 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     2041 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2101 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     2161 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     2221 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     2281 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     2341 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     2401 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     2461 aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag
     2521 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     2581 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     2641 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     2701 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     2761 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     2821 gcctaggctt ttgcaaaaag ctcccgcatg accgagtaca agcccacggt gcgcctcgcc
     2881 acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc
     2941 gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa
     3001 ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc
     3061 gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc
     3121 ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc
     3181 ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg
     3241 cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc
     3301 gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac
     3361 gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg
     3421 tgcatgaccc gcaagcccgg tgcctgattc gaaatgaccg accaagcgac gcccaacctg
     3481 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     3541 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     3601 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     3661 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     3721 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     3781 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     3841 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     3901 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     3961 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4021 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4081 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4141 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4201 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4261 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4321 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4381 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4441 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4501 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4561 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     4621 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4681 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4741 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4801 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     4861 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4921 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4981 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5041 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5101 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5161 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5221 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     5281 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5341 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5401 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5461 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5521 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     5581 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     5641 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     5701 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     5761 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     5821 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     5881 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacatt
     5941 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
     6001 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
     6061 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
     6121 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     6181 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
     6241 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
     6301 tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc
     6361 cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg
     6421 cggggggggg gggggsgcgc crggsggggs ggggsggggs grggggsggg gsggggsgag
     6481 gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc
     6541 gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc
     6601 gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg
     6661 actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa
     6721 ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa gccttgaggg
     6781 gctccgggag ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg tgtgtgtgtg
     6841 cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct gcgggcgcgg
     6901 cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg gcggtgcccc
     6961 gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt gcgtgggggg
     7021 gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc cccctccccg
     7081 agttgctgag cacggcccgg cttcgggtgc ggggctccgt acggggcgtg gcgcggggct
     7141 cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg ggccgcctcg
     7201 ggccggggag ggctcggggg aggggcgcgg cggcccccgg agcgccggcg gctgtcgagg
     7261 cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc gagagggcgc agggacttcc
     7321 tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc cctctagcgg
     7381 gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg gccttcgtgc
     7441 gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc ggggggacgg
     7501 ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga ccggcggctc
     7561 tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagctcc tgggcaacgt
     7621 gctggttatt gtgctgtctc atcattttgg caaagaattc ctcgaggaat tcactcctca
     7681 ggtgcaggct gcctatcaga aggtggtggc tggtgtggcc aatgccctgg ctcacaaata
     7741 ccactgagat ctgcatga
//
