LOCUS       sequence-360082-        4128 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CreOFF knock-in for GluA1-GFP (amino acid position: stop codon).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4128)
  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 4128)
  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 Gria1-GFP KI
FEATURES             Location/Qualifiers
     source          1..4128
                     /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             join(634..636,637..1350)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(634..636,637..1347)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(676..697)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(937..956)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1284..1305
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     repeat_region   1429..1569
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1429..1558
                     /label=AAV2 ITR
     rep_origin      1644..2099
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1731..1750)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1941..1962
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2116..2135)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2235..2257
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2295..2313)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2381..2485
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2486..2554,2555..3346)
                     /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(2704..2723)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3517..4105
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4006..4025
                     /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 ggggtgagca agggcgagga gctgttcacc
      661 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
      721 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
      781 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
      841 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
      901 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
      961 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     1021 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     1081 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     1141 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     1201 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     1261 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     1321 actctcggca tggacgagct gtacaagtaa ggcctcccgt tacaatcctg tggctcccct
     1381 cgaggatcct aggacgcgtt aacttacggt aaatggcccg gcggccgcag gaacccctag
     1441 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa
     1501 aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct
     1561 gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     1621 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     1681 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc
     1741 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     1801 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt
     1861 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     1921 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat
     1981 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa
     2041 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt
     2101 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     2161 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     2221 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     2281 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     2341 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     2401 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     2461 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     2521 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     2581 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     2641 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     2701 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     2761 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     2821 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     2881 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     2941 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     3001 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     3061 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     3121 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     3181 atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa
     3241 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     3301 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     3361 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     3421 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     3481 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     3541 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     3601 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     3661 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     3721 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     3781 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     3841 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3901 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3961 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     4021 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     4081 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gccttttt
//
