LOCUS       sequence-254361-        7938 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express Mac_Q139D_D150N_E258V-HaloTag in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7938)
  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 7938)
  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-Mac_Q139D_D150N_E258V-HaloTag
FEATURES             Location/Qualifiers
     source          1..7938
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1970..1987)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1976..2200
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2246..2674
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2333..2352)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2543..2564
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2683..2703)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2688..3017
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2868..3003
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2930..2949
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3028..3627
                     /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(3028..3047)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3524..3544
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3787..3908
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3824..3843)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3878..3897
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3957..3973)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3957..3973)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3970..3992)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3981..3997
                     /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(4005..4011,4012..4029,4030..4035))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4050..4071
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4188..4205)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4359..4947)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4439..4458)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5118..5909,5910..5978))
                     /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     5741..5760
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5979..6083)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        6115..6494
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          6770..7786
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     7710..7732
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     7794..7813
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
ORIGIN
        1 gctagccgcc accatgatcg ttgatcagtt cgaggaggtt ctcatgaaaa catcacagct
       61 cttccctctg ccaactgcaa cacaatctgc ccaacctacc catgttgcac ctgtaccgac
      121 cgtccttcct gacacaccga tttacgagac cgtcggagat agtggttcca aaactctttg
      181 ggtagtattc gtgttgatgc tcatagcatc cgcagctttc acggccttga gctggaagat
      241 acctgtaaac cggaggttgt accacgtaat caccactatc atcacactca cagccgcact
      301 gtcttatttt gccatggcaa ccggccatgg agtcgccctc aacaagatag tcatcaggac
      361 gcaacacgac catgtgccgg acacctatga aaccgtatat aggcaggtat actacgcacg
      421 gtacattgat tgggcaataa caaccccatt gcttctgctg aatctcgggt tgcttgcagg
      481 gatgtcagga gcacacattt ttatggcaat tgttgccgat cttattatgg tcttgacggg
      541 attgttcgct gcctttggca gtgaagggac tcctcaaaaa tggggctggt atacgattgc
      601 ttgcatcgcc tacatctttg ttgtctggca tctcgtactt aacggcggtg ctaacgccag
      661 ggttaaaggg gagaagcttc ggtctttttt cgttgctatc ggagcctaca ccctcatctt
      721 gtggacagct tatcctatcg tttggggcct ggccgacggg gctcgcaaaa ttggcgtaga
      781 cggggtgatt attgcctatg cagttctgga tgtactggct aaaggggtct tcggtgcatg
      841 gctcctggtc actcatgcca atcttcgcga atcagacatc ggtaccggct ttccattcga
      901 cccccattat gtggaagtcc tgggcgagcg catgcactac gtcgatgttg gtccgcgcga
      961 tggcacccct gtgctgttcc tgcacggtaa cccgacctcc tcctacgtgt ggcgcaacat
     1021 catcccgcat gttgcaccga cccatcgctg cattgctcca gacctgatcg gtatgggcaa
     1081 atccgacaaa ccagacctgg gttatttctt cgacgaccac gtccgcttca tggatgcctt
     1141 catcgaagcc ctgggtctgg aagaggtcgt cctggtcatt cacgactggg gctccgctct
     1201 gggtttccac tgggccaagc gcaatccaga gcgcgtcaaa ggtattgcat ttatggagtt
     1261 catccgccct atcccgacct gggacgaatg gccagaattt gcccgcgaga ccttccaggc
     1321 cttccgcacc accgacgtcg gccgcaagct gatcatcgat cagaacgttt ttatcgaggg
     1381 tacgctgccg atgggtgtcg tccgcccgct gactgaagtc gagatggacc attaccgcga
     1441 gccgttcctg aatcctgttg accgcgagcc actgtggcgc ttcccaaacg agctgccaat
     1501 cgccggtgag ccagcgaaca tcgtcgcgct ggtcgaagaa tacatggact ggctgcacca
     1561 gtcccctgtc ccgaagctgc tgttctgggg caccccaggc gttctgatcc caccggccga
     1621 agccgctcgc ctggccaaaa gcctgcctaa ctgcaaggct gtggacatcg gcccgggtct
     1681 gaatctgctg caagaagaca acccggacct gatcggcagc gagatcgcgc gctggctgtc
     1741 gacgctcgag atttccggcg agccaaccac taagagcagg atcaccagcg agggcgagta
     1801 catccccctg gaccagatcg acatcaacgt gttctgctac gagaacgagg tgtaatgaaa
     1861 gcttgctacc gagctcggat ccactagtcc agtgtggtgg aattctgcag atatccagca
     1921 cagtggcggc cgctcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg
     1981 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     2041 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     2101 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     2161 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     2221 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2281 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     2341 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     2401 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     2461 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     2521 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     2581 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     2641 aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag
     2701 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     2761 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     2821 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     2881 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     2941 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     3001 gcctaggctt ttgcaaaaag ctcccgcatg accgagtaca agcccacggt gcgcctcgcc
     3061 acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc
     3121 gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa
     3181 ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc
     3241 gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc
     3301 ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc
     3361 ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg
     3421 cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc
     3481 gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac
     3541 gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg
     3601 tgcatgaccc gcaagcccgg tgcctgattc gaaatgaccg accaagcgac gcccaacctg
     3661 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     3721 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     3781 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     3841 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     3901 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     3961 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     4021 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     4081 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4141 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4201 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4261 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4321 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4381 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4441 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4501 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4561 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4621 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4681 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4741 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     4801 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4861 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4921 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4981 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5041 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5101 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5161 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5221 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5281 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5341 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5401 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     5461 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5521 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5581 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5641 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5701 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     5761 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     5821 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     5881 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     5941 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6001 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6061 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacatt
     6121 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
     6181 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
     6241 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
     6301 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     6361 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
     6421 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
     6481 tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc
     6541 cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg
     6601 cggggggggg gggggsgcgc crggsggggs ggggsggggs grggggsggg gsggggsgag
     6661 gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc
     6721 gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc
     6781 gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg
     6841 actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa
     6901 ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa gccttgaggg
     6961 gctccgggag ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg tgtgtgtgtg
     7021 cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct gcgggcgcgg
     7081 cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg gcggtgcccc
     7141 gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt gcgtgggggg
     7201 gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc cccctccccg
     7261 agttgctgag cacggcccgg cttcgggtgc ggggctccgt acggggcgtg gcgcggggct
     7321 cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg ggccgcctcg
     7381 ggccggggag ggctcggggg aggggcgcgg cggcccccgg agcgccggcg gctgtcgagg
     7441 cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc gagagggcgc agggacttcc
     7501 tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc cctctagcgg
     7561 gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg gccttcgtgc
     7621 gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc ggggggacgg
     7681 ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga ccggcggctc
     7741 tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagctcc tgggcaacgt
     7801 gctggttatt gtgctgtctc atcattttgg caaagaattc ctcgaggaat tcactcctca
     7861 ggtgcaggct gcctatcaga aggtggtggc tggtgtggcc aatgccctgg ctcacaaata
     7921 ccactgagat ctgcatga
//
