LOCUS       sequence-251564-        4885 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  minicircle parental backbone plasmid including GFP knock-in donor 
            targeting Tubb3 gene and one cutting site for HITI .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4885)
  AUTHORS   Suzuki K, Tsunekawa Y, Hernandez-Benitez R, Wu J, Zhu J, Kim EJ, 
            Hatanaka F, Yamamoto M, Araoka T, Li Z, Kurita M, Hishida T, Li M, 
            Aizawa E, Guo S, Chen S, Goebl A, Soligalla RD, Qu J, Jiang T, Fu X,
            Jafari M, Esteban CR, Berggren WT, Lajara J, Nunez-Delicado E, 
            Guillen P, Campistol JM, Matsuzaki F, Liu GH, Magistretti P, Zhang 
            K, Callaway EM, Zhang K, Belmonte JC
  TITLE     In vivo genome editing via CRISPR/Cas9 mediated homology-independent
            targeted integration.
  JOURNAL   Nature. 2016 Dec 1;540(7631):144-149. doi: 10.1038/nature20565. Epub
            2016 Nov 16.
  PUBMED    27851729
REFERENCE   2  (bases 1 to 4885)
  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: 
            "10.1038/nature20565"; journalName: "Nature"; date: "2016-12-1- 1"; 
            volume: "540"; issue: "7631"; pages: "144-149"
COMMENT     Sequence Label: pTubb3-MC
FEATURES             Location/Qualifiers
     source          1..4885
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(743..760)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    781..814
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     CDS             join(862..864,865..1575)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(862..864,865..1575)
                     /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(904..925)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1165..1184)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1512..1533
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             complement(1708..2502)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(1708..2502)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1819..1838)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2429..2448
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(2866..2883)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3036..3122
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3214..3241
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      3375..3963
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3864..3883
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4117..4134
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4149..4289
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4215..4234)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4375..4397
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 acattaccct gttatcccta gatacattac cctgttatcc cagatgacat accctgttat
       61 ccctagatga cattaccctg ttatcccaga tgacattacc ctgttatccc tagatacatt
      121 accctgttat cccagatgac ataccctgtt atccctagat gacattaccc tgttatccca
      181 gatgacatta ccctgttatc cctagataca ttaccctgtt atcccagatg acataccctg
      241 ttatccctag atgacattac cctgttatcc cagatgacat taccctgtta tccctagata
      301 cattaccctg ttatcccaga tgacataccc tgttatccct agatgacatt accctgttat
      361 cccagatgac attaccctgt tatccctaga tacattaccc tgttatccca gatgacatac
      421 cctgttatcc ctagatgaca ttaccctgtt atcccagatg acattaccct gttatcccta
      481 gatacattac cctgttatcc cagatgacat accctgttat ccctagatga cattaccctg
      541 ttatcccaga tgacattacc ctgttatccc tagatacatt accctgttat cccagatgac
      601 ataccctgtt atccctagat gacattaccc tgttatccca gatgacatta ccctgttatc
      661 cctagataca ttaccctgtt atcccagatg acataccctg ttatccctag atgacattac
      721 cctgttatcc cagataaact caatgatgat gatgatgatg gtcgagactc agcggccgcg
      781 gtgccagggc gtgcccttgg gctccccggg cgcgactata agctgcgagc aacttcactt
      841 gggtatgccg gcggtagcgc tgtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
      901 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
      961 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     1021 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     1081 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     1141 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     1201 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     1261 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     1321 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     1381 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     1441 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
     1501 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     1561 gacgagctgt acaaggggcc cgccccaact ggggtaacct ttgagttctc tcagttgggg
     1621 gtaatcagca tcatgatgtg gtaccacatc atgatgctga ttataagaat gcggccgcca
     1681 cactctagtg gatctcgagt taataattca gaagaactcg tcaagaaggc gatagaaggc
     1741 gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc
     1801 gccgccaagc tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc
     1861 cacacccagc cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt
     1921 cggcaagcag gcatcgccat gggtcacgac gagatcctcg ccgtcgggca tgctcgcctt
     1981 gagcctggcg aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg
     2041 atcgacaaga ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg
     2101 gtcgaatggg caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat
     2161 ggatactttc tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc
     2221 caatagcagc cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac
     2281 gcccgtcgtg gccagccacg atagccgcgc tgcctcgtct tgcagttcat tcagggcacc
     2341 ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc
     2401 ggcatcagag cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca
     2461 agcggccgga gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc
     2521 tgtctcttga tcagagcttg atcccctgcg ccatcagatc cttggcggca agaaagccat
     2581 ccagtttact ttgcagggct tcccaacctt accagagggc gccccagctg gcaattccgg
     2641 ttcgcttgct gtccataaaa ccgcccagtc tagctatcgc catgtaagcc cactgcaagc
     2701 tacctgcttt ctctttgcgc ttgcgttttc ccttgtccag atagcccagt agctgacatt
     2761 catccggggt cagcaccgtt tctgcggact ggctttctac gcgctcgagg gggggccaaa
     2821 cggtctccag cttggctgtt ttggcggatg agagaagatt ttcagcctga tacagattaa
     2881 atcagaacgc agaagcggtc tgataaaaca gaatttgcct ggcggcagta gcgcggtggt
     2941 cccacctgac cccatgccga actcagaagt gaaacgccgt agcgccgatg gtagtgtggg
     3001 gtctccccat gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga
     3061 aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa
     3121 atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac
     3181 gcccgccata aactgccagg catcaaatta agcagaaggc catcctgacg gatggccttt
     3241 ttgcgtttct acaaactctt ttgtttattt ttctaaatac attcaaatat gtatccgctc
     3301 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     3361 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     3421 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     3481 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     3541 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     3601 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     3661 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     3721 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     3781 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     3841 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     3901 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     3961 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     4021 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     4081 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     4141 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     4201 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     4261 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     4321 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     4381 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag cagatcaatt
     4441 cgcgcgcgaa ggcgaagcgg catgcataat gtgcctgtca aatggacgaa gcagggattc
     4501 tgcaaaccct atgctactcc gtcaagccgt caattgtctg attcgttacc aattatgaca
     4561 acttgacggc tacatcattc actttttctt cacaaccggc acggaactcg ctcgggctgg
     4621 ccccggtgca ttttttaaat acccgcgaga aatagagttg atcgtcaaaa ccaacattgc
     4681 gaccgacggt ggcgataggc atccgggtgg tgctcaaaag cagcttcgcc tggctgatac
     4741 gttggtcctc gcgccagctt aagacgctaa tccctaactg ctggcggaaa agatgtgaca
     4801 gacgcgacgg cgacaagcaa acatgctgtg cgacgctggc gattcttctc tcatccgcca
     4861 aaacagccaa gctggagacc gtttg
//
