LOCUS       sequence-493924-        7448 bp    DNA     circular SYN 25-NOV-2025
DEFINITION  HaloTag labeled PINK1-SPARK.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7448)
  AUTHORS   Vineall KG, Andrikopoulos A, Sun MJ, Yan A, Hartanto ER, Schmitt DL
  TITLE     Visualizing PINK1 Activity Dynamics in Single Cells with a Phase 
            Separation-Based Kinase Activity Reporter
  JOURNAL   bioRxiv 2025.10.08.681260
REFERENCE   2  (bases 1 to 7448)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 25, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2025.10.08.681260"
COMMENT     Sequence Label: Halo-PINK1-SPARK
FEATURES             Location/Qualifiers
     source          1..7448
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             925..957
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(961..969,970..984)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             2197..2250
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     promoter        complement(3001..3019)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3002..3019)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3039..3056)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3045..3269
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3315..3743
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3402..3421)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3612..3633
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3752..3772)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3757..4086
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3937..4072
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3999..4018
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4153..4947
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4207..4226)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4817..4836
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5121..5242
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5158..5177)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5212..5231
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5291..5307)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5291..5307)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5304..5326)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5315..5331
                     /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(5339..5345,5346..5363,5364..5369))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5384..5405
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5522..5539)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5693..6281)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5773..5792)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6452..7243,7244..7312))
                     /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     7075..7094
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7313..7417)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgcggcc
      901 gccaccatgg tgcggggttc tggtatggct agcatgactg gtggacagca aatgggtcgg
      961 gatctgtacg acgatgacga taaggatccc atgggcgggg gcggtagtac attgtcagac
     1021 tataacatac agaaagagtc tactctccac ctcgtactgc ggggtgggct ggagtccggg
     1081 cttcggagcg gctccggggg aggggatata atggcggaaa tcggcaccgg ctttcctttt
     1141 gacccccact atgtggaagt gttgggtgag cggatgcact acgtggacgt gggacccaga
     1201 gatggcacac ccgtgctgtt ccttcatggc aacccgacgt cctcatatgt gtggcggaac
     1261 attattcctc atgtggctcc aactcataga acgatcgccc cagatctcat agggatgggg
     1321 aagagcgata agcctgatct ggggtacttt tttgacgacc atgtcagatt tatggacgcg
     1381 tttatcgaag cactgggatt ggaagaagtg gttctggtaa tccacgactg gggtagtgct
     1441 ctggggttcc actgggcaaa aaggaacccc gagcgcgtta aagggatcgc cttcatggaa
     1501 ttcatccgcc ctattccaac atgggatgag tggcctgagt tcgctcggga aacttttcag
     1561 gcttttcgga ccaccgatgt cggccggaaa ttgatcattg atcagaacgt attcatcgaa
     1621 ggcacactcc ctatgggcgt ggtgaggcct ctcacagagg tggaaatgga ccattacaga
     1681 gagccctttc ttaaccctgt cgacagagaa cctctctggc ggtttcctaa cgagcttcca
     1741 atcgctggcg aacctgctaa cattgtcgcc ctggtcgaag agtacatgga ctggttgcac
     1801 cagagccctg tgccaaaact gctgttttgg ggtaccccag gtgtgctcat tccccccgca
     1861 gaggctgcta gactggcaaa gtcccttccc aacgtcaaag ccgtggatat cggtcctggg
     1921 ctgaatttgt tgcaggagga taacccggac cttattgggt ccgagattgc aaggtggctg
     1981 tcaaccctgg aaatctcagg aggctctggg tccgcaggag ggagcgcagg cggttctgct
     2041 ggaggctcag caggcggctc cgccggcggg tctgcaggcg gaagtgctgg tgggtctcgc
     2101 ggagaaatcg cgaagtctct taaagaaatc gccaagtccc tcaaagaaat cgcttggtcc
     2161 ctgaaggaga ttgccaagtc ccttaagggg ggaactgagg gcaggggttc tctcctgacg
     2221 tgtggggacg tagaagagaa ccccggaccc aaactcgcca caatgcacgg caatggtagg
     2281 ttcctgaccc ttaagcctct gccggactca ataatccagg aaagtctgga gatccagcag
     2341 ggcgttaatc ctttcttcat cgggcggagt gaagactgta actgcaaaat cgaggattct
     2401 agactcagca gagtacactg cttcatattc aaaaagcgcc acgcagttgg aaagagcatg
     2461 tacgagagcc ctgcacaagg actcgatgat atttggtact gtcacaccgg tacaaatgtc
     2521 tcttatctca ataacaatag gatgatacag gggacaaagt ttcttctcca ggacggtgat
     2581 gagataaaaa ttatctggga caagaacaac aagtttgtga ttgggttcaa ggtcgagatc
     2641 aacgatacaa ccggcctgtt caatgaaggg cttgggatgc tgcaggaaca gcgggtagtg
     2701 cttaagcaga ctgccgaaga aaaggatctt gtaaaaaaac tcggatccgg ctccgcggga
     2761 ggatcagcag gcggcagcgc cgggggatct gctggcggta gtgccggcgg gagcgctggg
     2821 ggttcagcgg ggggatctag aaccctcagg gaaatcgagg agcttctccg gaagattatt
     2881 gaagacagcg tgaggtccgt tgctgagctg gaagacatcg aaaaatggct taaaaaaatc
     2941 gaattctgca gatatccatc acactggcgg ccgctcgagc atgcatctag agggccctat
     3001 tctatagtgt cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc cttctagttg
     3061 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     3121 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     3181 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     3241 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     3301 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     3361 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     3421 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     3481 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     3541 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3601 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     3661 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     3721 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     3781 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3841 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3901 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     3961 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     4021 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     4081 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga
     4141 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     4201 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     4261 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     4321 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     4381 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     4441 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     4501 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     4561 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     4621 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     4681 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     4741 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     4801 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     4861 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     4921 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     4981 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     5041 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     5101 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     5161 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     5221 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     5281 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     5341 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     5401 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5461 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     5521 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     5581 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5641 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5701 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5761 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5821 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5881 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5941 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     6001 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     6061 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6121 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6181 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6241 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6301 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6361 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     6421 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     6481 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     6541 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6601 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     6661 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     6721 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     6781 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     6841 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6901 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     6961 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     7021 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     7081 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     7141 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     7201 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     7261 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     7321 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     7381 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     7441 ctgacgtc
//
