LOCUS       sequence-360129-        4147 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CreOFF knock-in for GFP-beta-actin (amino acid position: before 
            start codon).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4147)
  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 4147)
  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 GFP-Actb KI
FEATURES             Location/Qualifiers
     source          1..4147
                     /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             join(635..637,638..1348)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(635..637,638..1348)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(677..698)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(938..957)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1285..1306
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     repeat_region   1448..1588
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1448..1577
                     /label=AAV2 ITR
     rep_origin      1663..2118
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1750..1769)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1960..1981
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2135..2154)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2254..2276
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2314..2332)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2400..2504
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2505..2573,2574..3365)
                     /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(2723..2742)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3536..4124
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4025..4044
                     /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 cttgtttgtg tgccttgata gttcgccagt tttagagcta
      421 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
      481 gtgctttttt gttttagagc tagaaatagc aagttaaaat aaggctagtc cgtttttagc
      541 gcgtgcgcca attctgcaga caaatggctc tagaagcttt cgaagaccct agaccctggc
      601 gaactatcaa ggcacattcg ccatggctag cggagtgagc aagggcgagg agctgttcac
      661 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
      721 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
      781 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
      841 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
      901 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
      961 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     1021 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     1081 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     1141 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     1201 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     1261 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     1321 cactctcggc atggacgagc tgtacaagag tggaagcgct ccctggcgaa ctatcaaggc
     1381 acactcgggg tcttctcctc gaggatccta ggacgcgtta acttacggta aatggcccgg
     1441 cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc
     1501 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg
     1561 agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat
     1621 ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg
     1681 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct
     1741 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     1801 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     1861 accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     1921 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     1981 gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt
     2041 cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     2101 tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     2161 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     2221 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     2281 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     2341 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     2401 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     2461 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     2521 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     2581 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     2641 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     2701 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     2761 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     2821 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     2881 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     2941 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     3001 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     3061 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     3121 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     3181 gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag
     3241 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     3301 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     3361 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     3421 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     3481 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3541 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     3601 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3661 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     3721 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     3781 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     3841 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     3901 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     3961 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     4021 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     4081 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     4141 ccttttt
//
