LOCUS       sequence-254362-        7565 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express Ace2_D92N_E199V-mRuby3 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7565)
  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 7565)
  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
FEATURES             Location/Qualifiers
     source          1..7565
                     /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(1595..1612)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1601..1825
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1871..2299
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1958..1977)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2168..2189
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2308..2328)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2313..2642
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2493..2628
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2555..2574
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2653..3252
                     /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(2653..2672)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3149..3169
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3412..3533
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3449..3468)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3503..3522
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3582..3598)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3582..3598)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3595..3617)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3606..3622
                     /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(3630..3636,3637..3654,3655..3660))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3675..3696
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3813..3830)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3984..4572)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4064..4083)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4743..5534,5535..5603))
                     /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     5366..5385
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5604..5708)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        5740..6119
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          6397..7413
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     7337..7359
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     7421..7440
                     /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 cgaggtgtaa tgaaagcttg ctaccgagct
     1501 cggatccact agtccagtgt ggtggaattc tgcagaaatc cagcacagtg gcggccgctc
     1561 gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     1621 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     1681 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     1741 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     1801 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     1861 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     1921 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     1981 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     2041 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     2101 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     2161 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     2221 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     2281 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     2341 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     2401 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     2461 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     2521 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     2581 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     2641 aaaagctccc gcatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc
     2701 cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc
     2761 gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc
     2821 gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg
     2881 accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc
     2941 gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac
     3001 cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc
     3061 aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg
     3121 cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc
     3181 accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag
     3241 cccggtgcct gattcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
     3301 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     3361 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
     3421 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     3481 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     3541 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     3601 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     3661 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     3721 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     3781 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     3841 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     3901 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     3961 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4021 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4081 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4141 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     4201 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4261 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4321 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     4381 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     4441 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4501 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     4561 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     4621 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     4681 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     4741 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     4801 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     4861 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     4921 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     4981 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     5041 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     5101 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     5161 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     5221 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     5281 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     5341 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     5401 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     5461 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     5521 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     5581 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     5641 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     5701 gttccgcgca catttccccg aaaagtgcca cctgacgtcg acattgatta ttgactagtt
     5761 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
     5821 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     5881 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
     5941 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
     6001 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
     6061 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
     6121 tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc ccacccccaa
     6181 ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg ggggggggnn
     6241 ggsgcgccrg gsggggsggg gsggggsgrg gggsggggsg gggsgaggcg gagaggtgcg
     6301 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
     6361 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc
     6421 cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta
     6481 ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta gcgcttggtt
     6541 taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct ccgggagggc
     6601 cctttgtgcg gggggagcgg ctcggggggt gcgtgcgtgt gtgtgtgcgt ggggagcgcc
     6661 gcgtgcggct ccgcgctgcc cggcggctgt gagcgctgcg ggcgcggcgc ggggctttgt
     6721 gcgctccgca gtgtgcgcga ggggagcgcg gccgggggcg gtgccccgcg gtgcgggggg
     6781 ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg tgggggggtg agcagggggt
     6841 gtgggcgcgt cggtcgggct gcaacccccc ctgcaccccc ctccccgagt tgctgagcac
     6901 ggcccggctt cgggtgcggg gctccgtacg gggcgtggcg cggggctcgc cgtgccgggc
     6961 ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc cgcctcgggc cggggagggc
     7021 tcgggggagg ggcgcggcgg cccccggagc gccggcggct gtcgaggcgc ggcgagccgc
     7081 agccattgcc ttttatggta atcgtgcgag agggcgcagg gacttccttt gtcccaaatc
     7141 tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct ctagcgggcg cggggcgaag
     7201 cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc ttcgtgcgtc gccgcgccgc
     7261 cgtccccttc tccctctcca gcctcggggc tgtccgcggg gggacggctg ccttcggggg
     7321 ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag agcctctgct
     7381 aaccatgttc atgccttctt ctttttccta cagctcctgg gcaacgtgct ggttattgtg
     7441 ctgtctcatc attttggcaa agaattcctc gaggaattca ctcctcaggt gcaggctgcc
     7501 tatcagaagg tggtggctgg tgtggccaat gccctggctc acaaatacca ctgagatctg
     7561 catga
//
