LOCUS       sequence-254291-        7976 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express soma-localized Ace1_Q89D_D100N_E206V-HaloTag in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7976)
  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 7976)
  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-Ace1_Q89D_D100N_E206V-HaloTag-ST
FEATURES             Location/Qualifiers
     source          1..7976
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(2006..2023)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2012..2236
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2282..2710
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2369..2388)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2579..2600
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2719..2739)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2724..3053
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2904..3039
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2966..2985
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3064..3663
                     /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(3064..3083)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3560..3580
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3823..3944
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3860..3879)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3914..3933
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3993..4009)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3993..4009)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4006..4028)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4017..4033
                     /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(4041..4047,4048..4065,4066..4071))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4086..4107
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4224..4241)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4395..4983)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4475..4494)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5154..5945,5946..6014))
                     /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     5777..5796
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6015..6119)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        6151..6530
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          6808..7824
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     7748..7770
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     7832..7851
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
ORIGIN
        1 gctagccgcc accatgagca atccaaatcc ctttcagact actcttggca cggacgccca
       61 gtgggttgtc tttgccgtaa tggcactggc agccatagtg tttagtatag cagttcagtt
      121 tcggccactc ccgctgcgcc tcacttacta cgtcaacatt gctatttgta caatagccgc
      181 aacagcttac tatgccatgg ccgttaatgg cggagacaat aaaccgacag ctgggacggg
      241 ggcagacgaa cgccaggtta tttacgctcg ctatatcgac tgggtgttta ccacaccact
      301 cctcttgctt aatctggtgt tgttgactaa catgccagcc actatgatcg cttggattat
      361 gggagcagac atcgctatga tcgcttttgg aatcatcggt gctttcaccg tcggctctta
      421 caagtggttt tacttcgtag tggggtgcat aatgctcgca gtccttgctt gggggatgat
      481 aaaccccata ttcaaagagg aattgcagaa acacaaagag tacacagggg cctacacgac
      541 tctgctcata tatctcatag tcttgtgggt tatatatcca attgtctggg gtctcggagc
      601 aggcgggcac attataggag tggatgtagt gattatcgca atgggtgttc tggacctgct
      661 tgccaagccg ctttacgcta taggggttct cataacggtt gaggttgttt acggaaagat
      721 cggtaccggc tttccattcg acccccatta tgtggaagtc ctgggcgagc gcatgcacta
      781 cgtcgatgtt ggtccgcgcg atggcacccc tgtgctgttc ctgcacggta acccgacctc
      841 ctcctacgtg tggcgcaaca tcatcccgca tgttgcaccg acccatcgct gcattgctcc
      901 agacctgatc ggtatgggca aatccgacaa accagacctg ggttatttct tcgacgacca
      961 cgtccgcttc atggatgcct tcatcgaagc cctgggtctg gaagaggtcg tcctggtcat
     1021 tcacgactgg ggctccgctc tgggtttcca ctgggccaag cgcaatccag agcgcgtcaa
     1081 aggtattgca tttatggagt tcatccaccc tatcccgacc tgggacgaat ggccagaatt
     1141 tgcccgcgag accttccagg ccttccgcac caccgacgtc ggccgcaagc tgatcatcga
     1201 tcagaacgtt tttatcgagg gtacgctgcc gatgggtgtc gtccgcccgc tgactgaagt
     1261 cgagatggac cattaccgcg agccgttcct gaatcctgtt gaccgcgagc cactgtggcg
     1321 cttcccaaac gagctgccaa tcgccggtga gccagcgaac atcgtcgcgc tggtcgaaga
     1381 atacatggac tggctgcacc agtcccctgt cccgaagctg ctgttctggg gcaccccagg
     1441 cgttctgatc ccaccggccg aagccgctcg cctggccaaa agcctgccta actgcaaggc
     1501 tgtggacatc ggcccgggtc tgaatctgct gcaagaagac aacccggacc tgatcggcag
     1561 cgagatcgcg cgctggctgt cgacgctcga gatttccggc gagccaacca ctaagagcag
     1621 gatcaccagc gagggcgagt acatccccct ggaccagatc gacatcaacg tgttctgcta
     1681 cgagaacgag gtgcaaagtc agcctatcct gaacacaaag gaaatggctc cacagtctaa
     1741 gcctcccgaa gagcttgaga tgtccagtat gccaagtccc gtggctcccc tccctgccag
     1801 gactgaagga gtgattgaca tgaggagtat gtcatctatt gatagcttca tctcttgcgc
     1861 aacagatttc cccgaggcta ctcgattcta atgaaagctt gctaccgagc tcggatccac
     1921 tagtccagtg tggtggaatt ctgcagatat ccagcacagt ggcggccgct cgagtctaga
     1981 gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     2041 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     2101 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     2161 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     2221 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc
     2281 cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     2341 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     2401 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     2461 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     2521 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     2581 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     2641 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     2701 aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     2761 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     2821 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     2881 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     2941 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg
     3001 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc
     3061 cgcatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc
     3121 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg
     3181 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     3241 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     3301 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     3361 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag
     3421 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg
     3481 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc
     3541 ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     3601 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     3661 tgattcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     3721 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     3781 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
     3841 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     3901 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     3961 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     4021 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     4081 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     4141 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     4201 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4261 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4321 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4381 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4441 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4501 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4561 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4621 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4681 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4741 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4801 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     4861 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4921 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4981 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     5041 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     5101 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     5161 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     5221 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     5281 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     5341 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     5401 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     5461 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     5521 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     5581 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     5641 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     5701 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     5761 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     5821 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     5881 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     5941 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     6001 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     6061 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     6121 acatttcccc gaaaagtgcc acctgacgtc gacattgatt attgactagt tattaatagt
     6181 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
     6241 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
     6301 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
     6361 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
     6421 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
     6481 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtcgaggtga
     6541 gccccacgtt ctgcttcact ctccccatct cccccccctc cccaccccca attttgtatt
     6601 tatttatttt ttaattattt tgtgcagcga tgggggcggg gggggggggn nsgsgcgccr
     6661 ggsggggsgg ggsggggsgr ggggsggggs ggggsgaggc ggagaggtgc ggcggcagcc
     6721 aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc
     6781 tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc
     6841 cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag
     6901 gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg
     6961 cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc
     7021 ggggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc cgcgtgcggc
     7081 tccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc
     7141 agtgtgcgcg aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg gggctgcgag
     7201 gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg
     7261 tcggtcgggc tgcaaccccc cctgcacccc cctccccgag ttgctgagca cggcccggct
     7321 tcgggtgcgg ggctccgtac ggggcgtggc gcggggctcg ccgtgccggg cggggggtgg
     7381 cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg ccggggaggg ctcgggggag
     7441 gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg cggcgagccg cagccattgc
     7501 cttttatggt aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat ctgtgcggag
     7561 ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc gcggggcgaa gcggtgcggc
     7621 gccggcagga aggaaatggg cggggagggc cttcgtgcgt cgccgcgccg ccgtcccctt
     7681 ctccctctcc agcctcgggg ctgtccgcgg ggggacggct gccttcgggg gggacggggc
     7741 agggcggggt tcggcttctg gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt
     7801 catgccttct tctttttcct acagctcctg ggcaacgtgc tggttattgt gctgtctcat
     7861 cattttggca aagaattcct cgaggaattc actcctcagg tgcaggctgc ctatcagaag
     7921 gtggtggctg gtgtggccaa tgccctggct cacaaatacc actgagatct gcatga
//
