LOCUS       sequence-173610-        4761 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Customizable donor vector for CRISPR/Cas9-mediated 
            homology-independent knock-in system..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4761)
  AUTHORS   Katoh Y, Michisaka S, Nozaki S, Funabashi T, Hirano T, Takei R, 
            Nakayama K
  TITLE     Practical method for targeted disruption of cilia-related genes by 
            using CRISPR/Cas9-mediated homology-independent knock-in system.
  JOURNAL   Mol Biol Cell. 2017 Feb 8. pii: mbc.E17-01-0051. doi: 
            10.1091/mbc.E17-01-0051.
  PUBMED    28179459
REFERENCE   2  (bases 1 to 4761)
  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"; journalName: "Mol Biol 
            Cell. 2017 Feb 8. pii: mbc.E17-01-0051. doi: 
            10.1091/mbc.E17-01-0051."
COMMENT     Sequence Label: pDonor-tBFP-NLS-Neo
FEATURES             Location/Qualifiers
     source          1..4761
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        60..363
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        364..567
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     518..538
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             606..1304
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             1329..1349
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1353..1373
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1377..1397
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1527..1648
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1564..1583)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1618..1637
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1655..2110)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1792..1813)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2004..2023
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2137..2241
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2243..2600
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2243..2434
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2266..2286)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2451..2586
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2635..3429
                     /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             2635..3429
                     /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(2689..2708)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3299..3318
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3617..3636)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3661..3708
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4037..4625
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4526..4545
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttataat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
       61 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
      121 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
      181 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
      241 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
      301 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
      361 atggtgatgc ggttttggca gtacatcaat gggcttggat agcggtttga ctcacgggga
      421 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
      481 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
      541 cggtgggagg tctatataag cagagctggt ttagtgaacc gtcagatccg ctagcggtcg
      601 ccaccatgag cgagctgatt aaggagaaca tgcacatgaa gctgtacatg gagggcaccg
      661 tggacaacca tcacttcaag tgcacatccg agggcgaagg caagccctac gagggcaccc
      721 agaccatgag aatcaaggtg gtcgagggcg gccctctccc cttcgccttc gacatcctgg
      781 ctactagctt cctctacggc agcaagacct tcatcaacca cacccagggc atccccgact
      841 tcttcaagca gtccttccct gagggcttca catgggagag agtcaccaca tacgaggacg
      901 ggggcgtgct gaccgctacc caggacacca gcctccagga cggctgcctc atctacaacg
      961 tcaagatcag aggggtgaac ttcacatcca acggccctgt gatgcagaag aaaacactcg
     1021 gctgggaggc cttcaccgag acgctgtacc ccgctgacgg cggcctggaa ggcagaaacg
     1081 acatggccct gaagctcgtg ggcgggagcc atctgatcgc aaacgccaag accacatata
     1141 gatccaagaa acccgctaag aacctcaaga tgcctggcgt ctactatgtg gactacagac
     1201 tggaaagaat caaggaggcc aacaacgaga cctacgtcga gcagcacgag gtggcagtgg
     1261 ccagatactg cgacctccct agcaaactgg ggcacaagct taattccgga ctcagatctc
     1321 gagctgatcc aaaaaagaag agaaaggtag atccaaaaaa gaagagaaag gtagatccaa
     1381 aaaagaagag aaaggtagga tccaccggat ctagataact gatcataatc agccatacca
     1441 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     1501 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat
     1561 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     1621 gtttgtccaa actcatcaat gtatcttaac gcgtaaattg taagcgttaa tattttgtta
     1681 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     1741 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     1801 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     1861 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     1921 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     1981 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     2041 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     2101 cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     2161 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     2221 taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt
     2281 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     2341 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     2401 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     2461 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     2521 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     2581 gaggcctagg cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt
     2641 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     2701 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     2761 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac
     2821 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     2881 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     2941 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     3001 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     3061 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     3121 caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag
     3181 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     3241 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     3301 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     3361 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     3421 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     3481 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     3541 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     3601 ctagggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga
     3661 cggcaataaa aagacagaat aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg
     3721 ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg
     3781 cccgcgtttc ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg
     3841 cagccaacgt cggggcggca ggccctgcca tagcctcagg ttactcatat atactttaga
     3901 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     3961 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     4021 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     4081 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     4141 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     4201 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     4261 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     4321 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     4381 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     4441 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     4501 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     4561 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     4621 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     4681 acatgttctt tcctgcgtta tcccctgatt ctgcaccggg tcttcgagaa gaccttggat
     4741 aaccgtatta ccgccatgca t
//
