LOCUS       sequence-360210-        4666 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CreOFF knock-in for GluA1-GFP-FRB (amino acid position: stop codon).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4666)
  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 4666)
  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: pOC1 Gria1-GFP-FRB KI
FEATURES             Location/Qualifiers
     source          1..4666
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(309..342)
                     /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(573..606)
                     /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        636..711
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             join(864..866,867..1574)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELY"
     CDS             join(864..866,867..1574)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELY"
     primer_bind     complement(906..927)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1167..1186)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1514..1535
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1602..1880
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     repeat_region   1967..2107
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1967..2096
                     /label=AAV2 ITR
     rep_origin      2182..2637
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2269..2288)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2479..2500
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2654..2673)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2773..2795
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2833..2851)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2919..3023
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3024..3092,3093..3884)
                     /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(3242..3261)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4055..4643
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4544..4563
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acggttcctg gccttttgct ggccttttgc tcacatgtga tccgacgccg ccatctctag
       61 gcccgcgccg gccccctcgc acagacttgt gggagaagct cggctactcc cctgccccgg
      121 ttaatttgca tataatattt cctagtaact atagaggctt aatgtgcgat aaaagacaga
      181 taatctgttc tttttaatac tagctacatt ttacatgata ggcttggatt tctataagag
      241 atacaaatac taaattatta ttttaaaaaa cagcacactc gagaactcac cctaactgta
      301 aagttagtat aacttcgtat agtataaatt atacgaagtt ataagccttg ttttttgcga
      361 tttttgaatt cgttcctcag aggaactgac aagcacccta acatcctatt ggaggctcac
      421 tcacgttttt tctattttgt ttcttgacag cagagctcgt tgctcactgt atagctcagg
      481 ttggcctgac actgatgagg ttctccagtg actgcctcta cctacctact gggatgacag
      541 aggtgtacca ccaagccacg cccgggggat ccataacttc gtatagtata aattatacga
      601 agttataagc cttgtgggag ccacaggatt gtaacgtttt agagctagaa atagcaagtt
      661 aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttgtt
      721 ttagagctag aaatagcaag ttaaaataag gctagtccgt ttttagcgcg tgcgccaatt
      781 ctgcagacaa atggctctag aagcttaccc gttacaatcc tgtggctccc ggcgctagcg
      841 gtagcggctc cagtagatct ggggtgagca agggcgagga gctgttcacc ggggtggtgc
      901 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
      961 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     1021 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
     1081 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     1141 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     1201 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     1261 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     1321 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     1381 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     1441 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
     1501 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     1561 tggacgagct gtacagcgct tctggtggtg gtagctacgt aatcctctgg catgagatgt
     1621 ggcatgaagg cctggaagag gcatctcgtt tgtactttgg ggaaaggaac gtgaaaggca
     1681 tgtttgaggt gctggagccc ttgcatgcta tgatggaacg gggcccccag actctgaagg
     1741 aaacatcctt taatcaggcc tatggtcgag atttaatgga ggcccaagag tggtgcagga
     1801 agtacatgaa atcagggaat gtcaaggacc tcctccaagc ctgggacctc tattatcatg
     1861 tgttccgacg aatctcaaag tgagtcgact gctcccgtta caatcctgtg gctcccacgc
     1921 gtggatccta ggaccggtcg ggccatttac cgtaagttgc ggccgcagga acccctagtg
     1981 atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag
     2041 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc
     2101 ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     2161 atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     2221 tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt
     2281 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     2341 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg
     2401 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     2461 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct
     2521 cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg
     2581 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat
     2641 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     2701 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     2761 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     2821 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     2881 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     2941 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3001 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     3061 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     3121 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     3181 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     3241 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     3301 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     3361 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     3421 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     3481 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     3541 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     3601 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     3661 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     3721 ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca gatggtaagc
     3781 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     3841 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     3901 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     3961 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4021 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4081 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4141 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4201 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4261 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4321 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4381 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4441 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4501 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4561 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4621 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc cttttt
//
