LOCUS       sequence-360079-        4267 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CreOFF knock-in for PSD95-Halo (amino acid position: R721).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4267)
  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 4267)
  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 Dlg4-Halo KI
FEATURES             Location/Qualifiers
     source          1..4267
                     /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        409..484
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             612..1502
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIHPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     repeat_region   1568..1708
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1568..1697
                     /label=AAV2 ITR
     rep_origin      1783..2238
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1870..1889)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2080..2101
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2255..2274)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2374..2396
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2434..2452)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2520..2624
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2625..2693,2694..3485)
                     /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(2843..2862)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3656..4244
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4145..4164
                     /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 cttgtttgaa tcagagtctc tctcgggcgt tttagagcta
      421 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
      481 gtgctttttt gttttagagc tagaaatagc aagttaaaat aaggctagtc cgtttttagc
      541 gcgtgcgcca attctgcaga caaatggctc tagaagctta atcagagtct ctctcgggcg
      601 gggctagcgg aatggcagaa atcggtactg gctttccatt cgacccccat tatgtggaag
      661 tcctgggcga gcgcatgcac tacgtcgatg ttggtccgcg cgatggcacc cctgtgctgt
      721 tcctgcacgg taacccgacc tcctcctacg tgtggcgcaa catcatcccg catgttgcac
      781 cgacccatcg ctgcattgct ccagacctga tcggtatggg caaatccgac aaaccagacc
      841 tgggttattt cttcgacgac cacgtccgct tcatggatgc cttcatcgaa gccctgggtc
      901 tggaagaggt cgtcctggtc attcacgact ggggctccgc tctgggtttc cactgggcca
      961 agcgcaatcc agagcgcgtc aaaggtattg catttatgga gttcatccac cctatcccga
     1021 cctgggacga atggccagaa tttgcccgcg agaccttcca ggccttccgc accaccgacg
     1081 tcggccgcaa gctgatcatc gatcagaacg tttttatcga gggtacgctg ccgatgggtg
     1141 tcgtccgccc gctgactgaa gtcgagatgg accattaccg cgagccgttc ctgaatcctg
     1201 ttgaccgcga gccactgtgg cgcttcccaa acgagctgcc aatcgccggt gagccagcga
     1261 acatcgtcgc gctggtcgaa gaatacatgg actggctgca ccagtcccct gtcccgaagc
     1321 tgctgttctg gggcacccca ggcgttctga tcccaccggc cgaagccgct cgcctggcca
     1381 aaagcctgcc taactgcaag gctgtggaca tcggcccggg tctgaatctg ctgcaagaag
     1441 acaacccgga cctgatcggc agcgagatcg cgcgctggct gtccacgctc gagatttccg
     1501 gcagcgctaa atcagagtct ctctcgggcg ggacgcgtta acttacggta aatggcccgg
     1561 cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc
     1621 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg
     1681 agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat
     1741 ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg
     1801 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct
     1861 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     1921 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     1981 accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     2041 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     2101 gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt
     2161 cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     2221 tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     2281 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     2341 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     2401 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     2461 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     2521 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     2581 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     2641 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     2701 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     2761 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     2821 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     2881 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     2941 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     3001 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     3061 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     3121 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     3181 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     3241 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     3301 gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag
     3361 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     3421 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     3481 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     3541 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     3601 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3661 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     3721 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3781 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     3841 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     3901 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     3961 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4021 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     4081 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     4141 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     4201 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     4261 ccttttt
//
