LOCUS       sequence-360207-        4290 bp    DNA     circular SYN 15-APR-2025
DEFINITION  CreOFF knock-in for GluA1-Halo (amino acid position: stop codon).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4290)
  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 4290)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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 Gria1-Halo KI
FEATURES             Location/Qualifiers
     source          1..4290
                     /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        406..481
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             634..1524
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     repeat_region   1591..1731
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1591..1720
                     /label=AAV2 ITR
     rep_origin      1806..2261
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1893..1912)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2103..2124
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2278..2297)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2397..2419
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2457..2475)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2543..2647
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2648..2716,2717..3508)
                     /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(2866..2885)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3679..4267
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4168..4187
                     /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 cttgtgggag ccacaggatt gtaacgtttt agagctagaa
      421 atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
      481 cttttttgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt ttttagcgcg
      541 tgcgccaatt ctgcagacaa atggctctag aagcttaccc gttacaatcc tgtggctccc
      601 ggcgctagcg gtagcggctc cagtagatct gggatggcag aaatcggtac tggctttcca
      661 ttcgaccccc attatgtgga agtcctgggc gagcgcatgc actacgtcga tgttggtccg
      721 cgcgatggca cccctgtgct gttcctgcac ggtaacccga cctcctccta cgtgtggcgc
      781 aacatcatcc cgcatgttgc accgacccat cgctgcattg ctccagacct gatcggtatg
      841 ggcaaatccg acaaaccaga cctgggttat ttcttcgacg accacgtccg cttcatggat
      901 gccttcatcg aagccctggg tctggaagag gtcgtcctgg tcattcacga ctggggctcc
      961 gctctgggtt tccactgggc caagcgcaat ccagagcgcg tcaaaggtat tgcatttatg
     1021 gagttcatcc gccctatccc gacctgggac gaatggccag aatttgcccg cgagaccttc
     1081 caggccttcc gcaccaccga cgtcggccgc aagctgatca tcgatcagaa cgtttttatc
     1141 gagggtacgc tgccgatggg tgtcgtccgc ccgctgactg aagtcgagat ggaccattac
     1201 cgcgagccgt tcctgaatcc tgttgaccgc gagccactgt ggcgcttccc aaacgagctg
     1261 ccaatcgccg gtgagccagc gaacatcgtc gcgctggtcg aagaatacat ggactggctg
     1321 caccagtccc ctgtcccgaa gctgctgttc tggggcaccc caggcgttct gatcccaccg
     1381 gccgaagccg ctcgcctggc caaaagcctg cctaactgca aggctgtgga catcggcccg
     1441 ggtctgaatc tgctgcaaga agacaacccg gacctgatcg gcagcgagat cgcgcgctgg
     1501 ctgtccacgc tcgagatttc cggctaagcg ctcccgttac aatcctgtgg ctcccacgcg
     1561 ttaacttacg gtaaatggcc cggcggccgc aggaacccct agtgatggag ttggccactc
     1621 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg
     1681 gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga
     1741 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc
     1801 atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     1861 gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct
     1921 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     1981 atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag
     2041 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     2101 tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga
     2161 tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa
     2221 atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac
     2281 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc
     2341 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     2401 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct
     2461 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     2521 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     2581 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     2641 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     2701 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     2761 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     2821 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     2881 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     2941 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     3001 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     3061 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     3121 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     3181 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     3241 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     3301 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     3361 tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     3421 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     3481 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     3541 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     3601 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     3661 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     3721 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     3781 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     3841 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     3901 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     3961 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     4021 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     4081 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     4141 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     4201 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     4261 atggaaaaac gccagcaacg cggccttttt
//
