LOCUS       sequence-296060-        5814 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning template for AAV-based ORANGE knock-in constructs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5814)
  AUTHORS   Willems J, de Jong APH, Scheefhals N, Mertens E, Catsburg LAE, 
            Poorthuis RB, de Winter F, Verhaagen J, Meye FJ, MacGillavry HD
  TITLE     ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope 
            tagging of endogenous proteins in neurons.
  JOURNAL   PLoS Biol. 2020 Apr 10;18(4):e3000665. doi: 
            10.1371/journal.pbio.3000665.
  PUBMED    32275651
REFERENCE   2  (bases 1 to 5814)
  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"; journalName: "PLoS 
            Biol."; date: "2020-04-10"; volume: "18(4):e3000665. doi"; pages: " 
            10.1371/journal.pbio.3000665"
COMMENT     Sequence Label: pAAV MCS mCherry-KASH
FEATURES             Location/Qualifiers
     source          1..5814
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        237..1415
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          468..1406
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1363..1383
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1422..2132
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MQVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYE
                     GTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMN
                     FEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGAL
                     KGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYER
                     AEGRHSTGGMDELYK"
     primer_bind     complement(1570..1588)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1838..1857
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2449..2925
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2648..2667)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     rep_origin      complement(3247..3835)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3327..3346)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4006..4797,4798..4866))
                     /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     4629..4648
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4867..4971)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5039..5057
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5095..5117)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5217..5236
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(5303..5758)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5440..5461)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5652..5671
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cgctattacg ccaggctgca gggggggggg gggggttggc cactccctct ctgcgcgctc
       61 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
      121 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
      181 cagatcccat ggggcgcgcc gctagcggta ccggatccta cgtatctaga gtcgacggct
      241 ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag
      301 gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg
      361 tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag
      421 tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggta agtgccgtgt
      481 gtggttcccg cgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactt
      541 ccacctggct gcagtacgtg attcttgatc ccgagcttcg ggttggaagt gggtgggaga
      601 gttcgaggcc ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg
      661 ggcgctgggg ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg
      721 ataagtctct agccatttaa aatttttgat gacctgctgc gacgcttttt ttctggcaag
      781 atagtcttgt aaatgcgggc caagatctgc acactggtat ttcggttttt ggggccgcgg
      841 gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc gaggcggggc ctgcgagcgc
      901 ggccaccgag aatcggacgg gggtagtctc aagctggccg gcctgctctg gtgcctggcc
      961 tcgcgccgcc gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg gcaccagttg
     1021 cgtgagcgga aagatggccg cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc
     1081 ggcgctcggg agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct
     1141 cagccgtcgc ttcatgtgac tccacggagt accgggcgct gtccaggcac ctcgattagt
     1201 tctcgagctt ttggagtacg tcgtctttag gttgggggga ggggttttat gcgatggagt
     1261 ttccccacac tgagtgggtg gagactgaag ttaggccagc ttggcacttg atgtaattct
     1321 ccttggaatt tgcccttttt gagtttggat cttggttcat tctcaagcct cagacagtgg
     1381 ttcaaagttt ttttcttcca tttcaggtgt cgtgagccac catgcaagtg agcaagggcg
     1441 aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct
     1501 ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca
     1561 cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc
     1621 tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg
     1681 actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg
     1741 acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca
     1801 aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacta
     1861 tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga
     1921 tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct
     1981 acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca
     2041 tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc
     2101 actccaccgg cggcatggac gagctgtaca agtccggact cagatctcga gaggaggagg
     2161 aggagacaga cagcaggatg ccccacctcg acagccccgg cagctcccag ccgagacgct
     2221 ccttcctctc aagggtgatc agggcagcgc taccgttgca gctgcttctg ctgctgctgc
     2281 tgctcctggc ctgcctgcta cctgcctctg aagatgacta cagctgcacc caggccaaca
     2341 actttgcccg atccttctac cccatgctgc ggtacaccaa cgggccacct cccacctagg
     2401 aattcgatat caagcttatc gataccgagc gctgctcgag agatctacgg gtggcatccc
     2461 tgtgacccct ccccagtgcc tctcctggcc ctggaagttg ccactccagt gcccaccagc
     2521 cttgtcctaa taaaattaag ttgcatcatt ttgtctgact aggtgtcctt ctataatatt
     2581 atggggtgga ggggggtggt atggagcaag gggcaagttg ggaagacaac ctgtagggcc
     2641 tgcggggtct attgggaacc aagctggagt gcagtggcac aatcttggct cactgcaatc
     2701 tccgcctcct gggttcaagc gattctcctg cctcagcctc ccgagttgtt gggattccag
     2761 gcatgcatga ccaggctcag ctaatttttg tttttttggt agagacgggg tttcaccata
     2821 ttggccaggc tggtctccaa ctcctaatct caggtgatct acccaccttg gcctcccaaa
     2881 ttgctgggat tacaggcgtg aaccactgct cccttccctg tccttctgat tttgtaggta
     2941 accacgtgcg gaccgagcgg ccgatctgag gaacccctag tgatggagtt ggccactccc
     3001 tctctgcagc ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     3061 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     3121 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     3181 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     3241 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     3301 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     3361 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     3421 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     3481 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     3541 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     3601 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     3661 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     3721 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     3781 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     3841 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     3901 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     3961 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     4021 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     4081 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     4141 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     4201 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     4261 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     4321 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     4381 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     4441 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     4501 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     4561 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     4621 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     4681 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     4741 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     4801 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     4861 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     4921 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     4981 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
     5041 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg
     5101 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     5161 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc
     5221 atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt
     5281 aaggagaaaa taccgcatca ggaaattgta agcgttaata ttttgttaaa attcgcgtta
     5341 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     5401 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     5461 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     5521 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     5581 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     5641 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     5701 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc
     5761 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tctt
//
