LOCUS       sequence-487109-        4543 bp    DNA     circular SYN 24-SEP-2025
DEFINITION  AAV vector for SpCas9-mediated CRISPR KI of oScarlet at the 
            C-terminus of mouse alpha-syn.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4543)
  AUTHORS   Parra-Rivas LA, Sharma R, Rust TE, Bazick HO, Carlson-Stevermer J, 
            Zylka MJ, Ogawa Y, Roy S
  TITLE     Protocol for CRISPR-based manipulation and visualization of 
            endogenous alpha-synuclein in cultured mouse hippocampal neurons.
  JOURNAL   STAR Protoc. 2025 Jul 21;6(3):103945. doi: 
            10.1016/j.xpro.2025.103945.
  PUBMED    40700012
REFERENCE   2  (bases 1 to 4543)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 24, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "STAR 
            Protoc."; date: "2025-07-21"; volume: "6(3):103945. doi"; pages: " 
            10.1016/j.xpro.2025.103945"
COMMENT     Sequence Label: pLP857_alpha-syn:oScarlet KI donor
FEATURES             Location/Qualifiers
     source          1..4543
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        147..395
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     147..167
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     318..337
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(703..736)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     740..760
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     CDS             1036..1728
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="VSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWDRVMNFEDGGA
                     VTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKM
                     ALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHS
                     TGGMDELYK"
     primer_bind     complement(1166..1184)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     repeat_region   1806..1946
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1806..1935
                     /label=AAV2 ITR
     rep_origin      2021..2476
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2108..2127)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2318..2339
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2493..2512)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2612..2634
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2672..2690)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2758..2862
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2863..2931,2932..3723)
                     /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     complement(3081..3100)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3894..4482
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4383..4402
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgctc acatgtgagg gcctatttcc catgattcct tcatatttgc
      181 atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga
      241 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
      301 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
      361 tggctttata tatcttgtgg aaaggacgaa acaccgaggc ttcaggctca tagtctgttt
      421 aagagctaag ctggaaacag catagcaagt ttaaataagg ctagtccgtt atcaacttga
      481 aaaagtggca ccgagtcggt gcttttttga tccgacgcgc catctctagg cccgcgccgg
      541 ccccctcgca cggacttgtg ggagaagctc ggctactccc ctgccccggt taatttgcat
      601 ataatatttc ctagtaacta tagaggctta atgtgcgata aaagacagat aatctgttct
      661 ttttaatact agctacattt tacatgatag gcttggattt ctataacttc gtatagcata
      721 cattatacga agttataaac agcacaaaag gaaactcacc ctaactgtaa agtaattgtg
      781 tgttttgaga ctataagtat cccttggaga accaccttgt tggaaacacc ggcgatcgta
      841 atcacccgag tgtttgagag ctaagcagaa agctgcatag caagttcaaa taaggctagt
      901 ccgtacacaa cttgaaaaag tggcagccga gtcggctgct tttttctgca ggcgatcgta
      961 atcacccgag tgggcgctcg aggtggaggc ggttcaggcg gaggtggctc tggcggtggc
     1021 ggatcgccat gggcggtgag caagggcgag gcagtgatca aggagttcat gcggttcaag
     1081 gtgcacatgg agggctccat gaacggccac gagttcgaga tcgagggcga gggcgagggc
     1141 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
     1201 ttctcctggg acatcctgtc ccctcagttc atgtacggct ccagggcctt caccaagcac
     1261 cccgccgaca tccccgacta ctataagcag tccttccccg agggcttcaa gtgggaccgc
     1321 gtgatgaact tcgaggacgg cggcgccgtg accgtgaccc aggacacctc cctggaggac
     1381 ggcaccctga tctacaaggt gaagctccgc ggcaccaact tccctcctga cggccccgta
     1441 atgcagaaga aaacaatggg ctgggaagcg tccaccgagc ggttgtaccc cgaggacggc
     1501 gtgctgaagg gcgacattaa gatggccctg cgcctgaagg acggcgggcg ctacctggcg
     1561 gacttcaaga ccacctacaa ggccaagaag cccgtgcaga tgcccggcgc ctacaacgtg
     1621 gaccgcaagt tggacatcac ctcccacaac gaggactaca ccgtggtgga acagtacgaa
     1681 cgctccgagg gccgccactc caccggcggc atggacgagc tgtacaagta atcgagaatt
     1741 cgatatcata gctagctagc tagggatccg cgatcgtaat cacccgagtg ggctagaggg
     1801 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     1861 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     1921 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     1981 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     2041 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta
     2101 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2161 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2221 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     2281 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     2341 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     2401 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     2461 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     2521 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     2581 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     2641 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     2701 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     2761 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     2821 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     2881 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     2941 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     3001 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     3061 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     3121 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     3181 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     3241 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     3301 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     3361 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     3421 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     3481 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     3541 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     3601 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     3661 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     3721 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     3781 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     3841 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     3901 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     3961 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     4021 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     4081 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     4141 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     4201 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     4261 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     4321 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     4381 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     4441 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     4501 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt
//
