LOCUS       sequence-360137-        3343 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CreOFF gRNA expression for beta3-tubulin (amino acid position: stop 
            codon).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3343)
  AUTHORS   Droogers WJ, Willems J, MacGillavry HD, de Jong APH
  TITLE     Duplex Labeling and Manipulation of Neuronal Proteins Using 
            Sequential CRISPR/Cas9 Gene Editing.
  JOURNAL   eNeuro. 2022 Jul 18;9(4):ENEURO.0056-22.2022. doi: 
            10.1523/ENEURO.0056-22.2022.
  PUBMED    35851300
REFERENCE   2  (bases 1 to 3343)
  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: 
            "10.1523/ENEURO.0056-22"; journalName: "eNeuro"; date: "2022-07-18- 
            18"; volume: "9"; issue: "4"; pages: "ENEURO.0056-22.2022"
COMMENT     Sequence Label: pOC2 Tubb3 no donor
FEATURES             Location/Qualifiers
     source          1..3343
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(39..72)
                     /label=loxP511
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATAC) (Shaw et al., 2021). loxP511 sites are 
                     compatible with each other, but incompatible with wild-type
                     loxP sites."
     protein_bind    complement(343..376)
                     /label=loxP511
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATAC) (Shaw et al., 2021). loxP511 sites are 
                     compatible with each other, but incompatible with wild-type
                     loxP sites."
     misc_RNA        408..483
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     repeat_region   644..784
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   644..773
                     /label=AAV2 ITR
     rep_origin      859..1314
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(946..965)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1156..1177
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1331..1350)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1450..1472
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1510..1528)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1596..1700
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1701..1769,1770..2561)
                     /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(1919..1938)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2732..3320
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3221..3240
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acggttcctg gccttttgct ggccttttgc tcacatgtat aacttcgtat agtataaatt
       61 atacgaagtt atgatccgac gccgccatct ctaggcccgc gccggccccc tcgcacagac
      121 ttgtgggaga agctcggcta ctcccctgcc ccggttaatt tgcatataat atttcctagt
      181 aactatagag gcttaatgtg cgataaaaga cagataatct gttcttttta atactagcta
      241 cattttacat gataggcttg gatttctata agagatacaa atactaaatt attattttaa
      301 aaaacagcac actcgagaac tcaccctaac tgtaaagtta gtataacttc gtatagtata
      361 aattatacga agttataagc cttgtttggc tgcgagcaac ttcacttgtt ttagagctag
      421 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg
      481 tgcttttttg ttttagagct agaaatagca agttaaaata aggctagtcc gtttttagcg
      541 cgtgcgccaa ttctgcagac aaatggctct agaagcttgc tagcaattgt cgactcgagg
      601 atcctaggac gcgttaactt acggtaaatg gcccggcggc cgcaggaacc cctagtgatg
      661 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
      721 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg
      781 caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
      841 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
      901 ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct
      961 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     1021 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     1081 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     1141 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg
     1201 gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc
     1261 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     1321 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     1381 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     1441 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     1501 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     1561 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     1621 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     1681 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     1741 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     1801 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     1861 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     1921 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     1981 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     2041 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     2101 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     2161 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     2221 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     2281 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     2341 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     2401 gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct
     2461 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     2521 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     2581 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     2641 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     2701 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     2761 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     2821 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     2881 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     2941 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     3001 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3061 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     3121 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     3181 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     3241 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     3301 gggggcggag cctatggaaa aacgccagca acgcggcctt ttt
//
