LOCUS       sequence-493925-        7126 bp    DNA     circular SYN 25-NOV-2025
DEFINITION  PINK1-SPARK control construct with PINK1 phosphosite mutated.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7126)
  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 7126)
  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: PINK1-SPARK S/A
FEATURES             Location/Qualifiers
     source          1..7126
                     /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             join(1027..1029,1030..1032,1033..1743)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1072..1093)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1333..1352)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1680..1701
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1939..1992
                     /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"
     primer_bind     complement(2717..2734)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2723..2947
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2993..3421
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3080..3099)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3290..3311
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3430..3450)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3435..3764
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3615..3750
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3677..3696
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3831..4625
                     /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(3885..3904)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4495..4514
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4799..4920
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4836..4855)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4890..4909
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4969..4985)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4969..4985)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4982..5004)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4993..5009
                     /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(5017..5023,5024..5041,5042..5047))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5062..5083
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5200..5217)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5371..5959)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5451..5470)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6130..6921,6922..6990))
                     /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     6753..6772
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6991..7095)
                     /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 gctggctagc
      901 gccaccatgg gcggtggagg gagcaccctg agcgattata acattcagaa agaagccacc
      961 ctgattctgg tgctgcgcgg cggactcgag tccggactca gatctggcag cggtggaggc
     1021 gatatcatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     1081 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
     1141 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     1201 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
     1261 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     1321 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     1381 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     1441 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     1501 aagaacggca tcaaggccaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     1561 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     1621 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     1681 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     1741 aagggatccg gcagtgctgg tggtagtgct ggtggtagtg ctggtggtag tgctggtggc
     1801 agtgctggtg gtagtgctgg tggtagtgct ggtggctcta gaggagaaat cgcaaagagt
     1861 ctgaaagaaa ttgcaaaatc cttgaaggaa atagcgtggt cactgaaaga aattgccaag
     1921 tccctcaaag ggggtaccga gggcagagga agtctgctaa catgcggtga cgtcgaggag
     1981 aatcctggcc caaagcttgc caccatgcat ggtaatggta ggtttttaac tttaaaacca
     2041 ttgcctgaca gcattattca agaaagcctg gagattcagc aaggtgtgaa tccatttttc
     2101 attggtagat ccgaggattg caattgtaaa attgaagaca gtaggttgtc tcgagttcat
     2161 tgcttcattt tcaaaaagag gcatgctgta ggcaaaagca tgtatgaatc tccggcacaa
     2221 ggtttagatg atatttggta ttgccacacc ggaactaacg tgagctattt aaataataac
     2281 cgcatgatac agggtacgaa attcctttta caagacggag atgaaatcaa gatcatttgg
     2341 gataaaaaca ataaatttgt cattggcttt aaagtggaaa ttaacgatac tacaggtctg
     2401 tttaacgagg gattaggtat gttacaagaa caaagagtag tacttaagca aacagccgaa
     2461 gaaaaagatt tggtgaaaaa gttaggatcc ggcagtgctg gtggtagtgc tggtggcagt
     2521 gctggtggta gtgctggtgg cagtgctggt ggtagtgctg gtggtagtgc tggtggctct
     2581 agaaccttga gagaaatcga ggaattgttg aggaaaatca ttgaagacag cgtgcgcagt
     2641 gtggccgagc tggaggacat tgagaagtgg ctgaagaaaa tctaagaatt cgggcccgtt
     2701 taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     2761 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     2821 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     2881 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     2941 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
     3001 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     3061 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     3121 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     3181 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     3241 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     3301 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     3361 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     3421 taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     3481 gaagtatgca aagcatgtat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     3541 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     3601 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     3661 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc
     3721 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt
     3781 gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac
     3841 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     3901 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     3961 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg
     4021 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     4081 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     4141 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     4201 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     4261 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     4321 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc
     4381 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     4441 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     4501 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     4561 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     4621 tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg
     4681 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     4741 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     4801 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     4861 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     4921 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     4981 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     5041 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     5101 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5161 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     5221 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     5281 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5341 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5401 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5461 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5521 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5581 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5641 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5701 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5761 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     5821 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     5881 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5941 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6001 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6061 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6121 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6181 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6241 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     6301 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6361 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6421 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6481 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     6541 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     6601 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     6661 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     6721 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     6781 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     6841 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     6901 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     6961 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7021 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7081 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
