LOCUS       sequence-205349-        6050 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Donor plasmid for SNCA exon3 wild type sequence. lso contains TagBFP
            and dTomato.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 6050)
  AUTHORS   Arias-Fuenzalida J, Jarazo J, Qing X, Walter J, Gomez-Giro G, 
            Nickels SL, Zaehres H, Scholer HR, Schwamborn JC
  TITLE     FACS-Assisted CRISPR-Cas9 Genome Editing Facilitates Parkinson's 
            Disease Modeling.
  JOURNAL   Stem Cell Reports. 2017 Nov 14;9(5):1423-1431. doi: 
            10.1016/j.stemcr.2017.08.026. Epub 2017 Oct 5.
  PUBMED    28988985
REFERENCE   2  (bases 1 to 6050)
  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"; doi: 
            "10.1016/j.stemcr.2017.08.026"; journalName: "Stem Cell Reports"; 
            date: "2017-11-14- 14"; volume: "9"; issue: "5"; pages: "1423-1431"
COMMENT     Sequence Label: pDONOR-SNCAe3-WT
FEATURES             Location/Qualifiers
     source          1..6050
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             1..702
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLYGMDELYK"
     primer_bind     complement(134..152)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     402..421
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     596..619
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             703..756
                     /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"
     CDS             757..1353
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     1250..1270
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(1432..1451)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1497..1552
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1551..1570)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     repeat_region   2082..2144
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3143..3163)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3143..3161)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3182..3198)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3182..3198)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3195..3217)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3206..3222
                     /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(3230..3236,3237..3254,3255..3260))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3275..3296
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3413..3430)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3584..4172)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3664..3683)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4343..5134,5135..5203))
                     /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     4966..4985
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5204..5308)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5334..5789)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5471..5492)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5683..5702
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5916..5938
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5930..5947
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5931..5947
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5957..5976
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5957..5975
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 atggtgagca agggcgagga ggtcatcaaa gagttcatgc gcttcaaggt gcgcatggag
       61 ggctccatga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
      121 ggcacccaga ccgccaagct gaaggtgacc aagggcggcc ccctgccctt cgcctgggac
      181 atcctgtccc cccagttcat gtacggctcc aaggcgtacg tgaagcaccc cgccgacatc
      241 cccgattaca agaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
      301 gaggacggcg gtctggtgac cgtgacccag gactcctccc tgcaggacgg cacgctgatc
      361 tacaaggtga agatgcgcgg caccaacttc ccccccgacg gccccgtaat gcagaagaag
      421 accatgggct gggaggcctc caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc
      481 gagatccacc aggccctgaa gctgaaggac ggcggccact acctggtgga gttcaagacc
      541 atctacatgg ccaagaagcc cgtgcaactg cccggctact actacgtgga caccaagctg
      601 gacatcacct cccacaacga ggactacacc atcgtggaac agtacgagcg ctccgagggc
      661 cgccaccacc tgttcctgta cggcatggac gagctgtaca aggagggcag aggaagtctt
      721 ctaacatgcg gtgacgtgga ggagaatccc ggccctaccg agtacaagcc cacggtgcgc
      781 ctcgccaccc gcgacgacgt cccccgggcc gtacgcaccc tcgccgccgc gttcgccgac
      841 taccccgcca cgcgccacac cgtcgacccg gaccgccaca tcgagcgggt caccgagctg
      901 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
      961 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     1021 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     1081 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc
     1141 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     1201 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
     1261 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     1321 acctggtgca tgacccgcaa gcccggtgcc tgacactcct caggtgcagg ctgcctatca
     1381 gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa taccactgag atctttttcc
     1441 ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata
     1501 aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggaag
     1561 gacatatggg agggcaaatc atttaaaaca tcagaatgag tatttggttt agagtttggc
     1621 aacatatgcc catatgctgg ctgccatgaa caaaggttgg ctataaagag gtcatcagta
     1681 tatgaaacag ccccctgctg tccattcctt attccataga aaagccttga cttgaggtta
     1741 gatttttttt atattttgtt ttgtgttatt tttttcttta acatccctaa aattttcctt
     1801 acatgtttta ctagccagat ttttcctcct ctcctgacta ctcccagtca tagctgtccc
     1861 tcttctctta tggagatccc tcgacctgca gcccaagctt ggatccctcg acttttgtta
     1921 ctttatagaa gaaattttga gtttttgttt ttttttaata aataaataaa cataaataaa
     1981 ttgtttgttg aatttattat tagtatgtaa gtgtaaatat aataaaactt aatatctatt
     2041 caaattaata aataaacctc gatatacaga ccgataaaac acatgcgtca attttacgca
     2101 tgattatctt taacgtacgt cacaatatga ttatctttct agggttaaag tatctagtgg
     2161 ttagtgtggc ccagtattca agattcctat gaaattgtaa aacaatcact gagcattcta
     2221 agaacatatc agtcttattg aaactgaatt ctttataaag tatttttaaa taggtaaata
     2281 ttgattataa ataaaaaata tacttgccaa gaataatgag ggctttgaat tgataagcta
     2341 tgtttaattt atagtaagtg ggcatttaaa tattctgacc aaaaatgtat tgacaaactg
     2401 ctgacaaaaa taaaatgtga atattgccat aattttaaaa aaagtaaaat ttctgttgat
     2461 tacagtaaaa tattttgacc ttaaattatg ttgattacaa tattcctttg ataattcaga
     2521 gtgcatttca ggaaacaccc ttggacagtc agtaaattgt ttattgtatt tatctttgta
     2581 ttgttatggt atagctattt gtacaaatat tattgtgcaa ttattacatt tctgattata
     2641 ttattcattt ggcctaaatt taccgagaat ttgaacaagt caattaggtt tataatcaag
     2701 aaatatcaaa aatgatgaaa aggatgataa tcatcatcag atgttgagga agatgaggat
     2761 gagagtgcca gaaatagaga aatcaaagga gaaccaaaat ttaacaaatt aaaagcccac
     2821 agacttgctg taattaagtt ttctgttgta agtactccac gtttcctggc agatgtggtg
     2881 aagcaaaaga tataatcaga aatataattt atatgatcgg aaagcattaa acacaatagt
     2941 gcctatacaa ataaaatgtt cctatcactg acttctaaaa tggaaatgag gacaatgata
     3001 tgggaatctt aatacagtgt tgtggatagg actaaaaaca caggagtcag atcttcttgg
     3061 ttcaacttcc tgcttactcc ttaccagctg tgtgaaccta gtatacgcgt atgcagccgc
     3121 ttaattaatc cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat
     3181 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     3241 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     3301 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     3361 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     3421 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     3481 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     3541 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     3601 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     3661 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     3721 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     3781 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     3841 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     3901 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     3961 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     4021 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4081 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     4141 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     4201 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     4261 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     4321 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     4381 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     4441 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     4501 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     4561 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     4621 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     4681 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     4741 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     4801 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     4861 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     4921 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     4981 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     5041 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     5101 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     5161 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     5221 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     5281 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt
     5341 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     5401 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     5461 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     5521 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     5581 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     5641 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     5701 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     5761 cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg
     5821 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
     5881 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
     5941 ggccagtgag cgcgcgtaat acgactcact atagggcgaa ttggggcgcg ccattcattg
     6001 attattgact agttactaat agtaatcaat tacggggtca ttagttcata
//
