LOCUS       sequence-493921-        7448 bp    DNA     circular SYN 25-NOV-2025
DEFINITION  HaloTag labeled PINK1-SPARK control construct with PINK1 phosphosite
            mutated.
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 S/A
FEATURES             Location/Qualifiers
     source          1..7448
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /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     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1211..1230)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1402..1781
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1782..1985
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1936..1956
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2030..2049
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2030..2048
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2068..2077
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2092..2124
                     /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(2128..2136,2137..2151)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             3364..3417
                     /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(4168..4186)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4169..4186)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4206..4223)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4212..4436
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4482..4910
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     4779..4800
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4919..4939)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4924..5253
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5104..5239
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5166..5185
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5320..6114
                     /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(5374..5393)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5984..6003
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6288..6409
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6325..6344)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6379..6398
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6458..6474)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6458..6474)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6471..6493)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6482..6498
                     /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(6506..6512,6513..6530,6531..6536))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6551..6572
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6689..6706)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6860..7448)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6940..6959)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
     1201 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     1261 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
     1321 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
     1381 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
     1441 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     1501 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     1561 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     1621 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     1681 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
     1741 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
     1801 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     1861 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
     1921 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     1981 gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc
     2041 actataggga gacccaagct tgcggccgcc accatggtgc ggggttctgg tatggctagc
     2101 atgactggtg gacagcaaat gggtcgggat ctgtacgacg atgacgataa ggatcccatg
     2161 ggcgggggcg gtagtacatt gtcagactat aacatacaga aagaggctac tctcattctc
     2221 gtactgcggg gtgggctgga gtccgggctt cggagcggct ccgggggagg ggatataatg
     2281 gcggaaatcg gcaccggctt tccttttgac ccccactatg tggaagtgtt gggtgagcgg
     2341 atgcactacg tggacgtggg acccagagat ggcacacccg tgctgttcct tcatggcaac
     2401 ccgacgtcct catatgtgtg gcggaacatt attcctcatg tggctccaac tcatagaacg
     2461 atcgccccag atctcatagg gatggggaag agcgataagc ctgatctggg gtactttttt
     2521 gacgaccatg tcagatttat ggacgcgttt atcgaagcac tgggattgga agaagtggtt
     2581 ctggtaatcc acgactgggg tagtgctctg gggttccact gggcaaaaag gaaccccgag
     2641 cgcgttaaag ggatcgcctt catggaattc atccgcccta ttccaacatg ggatgagtgg
     2701 cctgagttcg ctcgggaaac ttttcaggct tttcggacca ccgatgtcgg ccggaaattg
     2761 atcattgatc agaacgtatt catcgaaggc acactcccta tgggcgtggt gaggcctctc
     2821 acagaggtgg aaatggacca ttacagagag ccctttctta accctgtcga cagagaacct
     2881 ctctggcggt ttcctaacga gcttccaatc gctggcgaac ctgctaacat tgtcgccctg
     2941 gtcgaagagt acatggactg gttgcaccag agccctgtgc caaaactgct gttttggggt
     3001 accccaggtg tgctcattcc ccccgcagag gctgctagac tggcaaagtc ccttcccaac
     3061 gtcaaagccg tggatatcgg tcctgggctg aatttgttgc aggaggataa cccggacctt
     3121 attgggtccg agattgcaag gtggctgtca accctggaaa tctcaggagg ctctgggtcc
     3181 gcaggaggga gcgcaggcgg ttctgctgga ggctcagcag gcggctccgc cggcgggtct
     3241 gcaggcggaa gtgctggtgg gtctcgcgga gaaatcgcga agtctcttaa agaaatcgcc
     3301 aagtccctca aagaaatcgc ttggtccctg aaggagattg ccaagtccct taagggggga
     3361 actgagggca ggggttctct cctgacgtgt ggggacgtag aagagaaccc cggacccaaa
     3421 ctcgccacaa tgcacggcaa tggtaggttc ctgaccctta agcctctgcc ggactcaata
     3481 atccaggaaa gtctggagat ccagcagggc gttaatcctt tcttcatcgg gcggagtgaa
     3541 gactgtaact gcaaaatcga ggattctaga ctcagcagag tacactgctt catattcaaa
     3601 aagcgccacg cagttggaaa gagcatgtac gagagccctg cacaaggact cgatgatatt
     3661 tggtactgtc acaccggtac aaatgtctct tatctcaata acaataggat gatacagggg
     3721 acaaagtttc ttctccagga cggtgatgag ataaaaatta tctgggacaa gaacaacaag
     3781 tttgtgattg ggttcaaggt cgagatcaac gatacaaccg gcctgttcaa tgaagggctt
     3841 gggatgctgc aggaacagcg ggtagtgctt aagcagactg ccgaagaaaa ggatcttgta
     3901 aaaaaactcg gatccggctc cgcgggagga tcagcaggcg gcagcgccgg gggatctgct
     3961 ggcggtagtg ccggcgggag cgctgggggt tcagcggggg gatctagaac cctcagggaa
     4021 atcgaggagc ttctccggaa gattattgaa gacagcgtga ggtccgttgc tgagctggaa
     4081 gacatcgaaa aatggcttaa aaaaatcgaa ttctgcagat atccatcaca ctggcggccg
     4141 ctcgagcatg catctagagg gccctattct atagtgtcac ctaaatgcta gagctcgctg
     4201 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     4261 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     4321 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     4381 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     4441 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     4501 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     4561 agcgcccgct cctttcgctt tcttcctttc ctttctcgcc acgttcgccg gctttccccg
     4621 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     4681 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     4741 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     4801 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     4861 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
     4921 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     4981 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     5041 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     5101 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     5161 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     5221 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
     5281 ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     5341 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     5401 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     5461 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
     5521 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     5581 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     5641 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     5701 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     5761 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     5821 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
     5881 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     5941 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     6001 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     6061 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     6121 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     6181 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     6241 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     6301 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     6361 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     6421 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     6481 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     6541 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     6601 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     6661 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     6721 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     6781 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     6841 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     6901 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     6961 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     7021 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     7081 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     7141 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     7201 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     7261 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     7321 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     7381 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     7441 gatctcaa
//
