LOCUS       sequence-355146-        4761 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Entry clone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4761)
  AUTHORS   Reint G, Li Z, Labun K, Keskitalo S, Soppa I, Mamia K, Tolo E, 
            Szymanska M, Meza-Zepeda LA, Lorenz S, Cieslar-Pobuda A, Hu X, 
            Bordin DL, Staerk J, Valen E, Schmierer B, Varjosalo M, Taipale J, 
            Haapaniemi E
  TITLE     Rapid genome editing by CRISPR-Cas9-POLD3 fusion.
  JOURNAL   Elife. 2021 Dec 13;10. pii: 75415. doi: 10.7554/eLife.75415.
  PUBMED    34898428
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: "Elife."; 
            date: "2021-12-13"; volume: "10. pii: 75415. doi"; pages: " 
            10.7554/eLife.75415"
COMMENT     Sequence Label: pDONR221 Cas9-RFC5
FEATURES             Location/Qualifiers
     source          1..4761
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    570..801
                     /gene="mutant version of attP"
                     /label=attP1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(1197..1502)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(1299..1318)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1822..2505)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     2420..2439
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2506..2608)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(2753..2984)
                     /gene="mutant version of attP"
                     /label=attP2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3002..3021)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3003..3021)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3026..3042)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3026..3042)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3144..3163)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3155..3964
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3222..3241)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4111..4699
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4600..4619
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agctgaacga gaaacgtaaa atgatataaa tatcaatata ttaaattaga ttttgcataa
      721 aaaacagact acataatact gtaaaacaca acatatccag tcactatgaa tcaactactt
      781 agatggtatt agtgacctgt agtcgaccga cagccttcca aatgttcttc gggtgatgct
      841 gccaacttag tcgaccgaca gccttccaaa tgttcttctc aaacggaatc gtcgtatcca
      901 gcctactcgc tattgtcctc aatgccgtat taaatcataa aaagaaataa gaaaaagagg
      961 tgcgagcctc ttttttgtgt gacaaaataa aaacatctac ctattcatat acgctagtgt
     1021 catagtcctg aaaatcatct gcatcaagaa caatttcaca actcttatac ttttctctta
     1081 caagtcgttc ggcttcatct ggattttcag cctctatact tactaaacgt gataaagttt
     1141 ctgtaatttc tactgtatcg acctgcagac tggctgtgta taagggagcc tgacatttat
     1201 attccccaga acatcaggtt aatggcgttt ttgatgtcat tttcgcggtg gctgagatca
     1261 gccacttctt ccccgataac ggagaccggc acactggcca tatcggtggt catcatgcgc
     1321 cagctttcat ccccgatatg caccaccggg taaagttcac gggagacttt atctgacagc
     1381 agacgtgcac tggccagggg gatcaccatc cgtcgcccgg gcgtgtcaat aatatcactc
     1441 tgtacatcca caaacagacg ataacggctc tctcttttat aggtgtaaac cttaaactgc
     1501 atttcaccag cccctgttct cgtcagcaaa agagccgttc atttcaataa accgggcgac
     1561 ctcagccatc ccttcctgat tttccgcttt ccagcgttcg gcacgcagac gacgggcttc
     1621 attctgcatg gttgtgctta ccagaccgga gatattgaca tcatatatgc cttgagcaac
     1681 tgatagctgt cgctgtcaac tgtcactgta atacgctgct tcatagcata cctctttttg
     1741 acatacttcg ggtatacata tcagtatata ttcttatacc gcaaaaatca gcgcgcaaat
     1801 acgcatactg ttatctggct tttagtaagc cggatccacg cggcgtttac gccccccctg
     1861 ccactcatcg cagtactgtt gtaattcatt aagcattctg ccgacatgga agccatcaca
     1921 aacggcatga tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt gcgtataata
     1981 tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa
     2041 actggtgaaa ctcacccagg gattggctga gacgaaaaac atattctcaa taaacccttt
     2101 agggaaatag gccaggtttt caccgtaaca cgccacatct tgcgaatata tgtgtagaaa
     2161 ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa aacgtttcag tttgctcatg
     2221 gaaaacggtg taacaagggt gaacactatc ccatatcacc agctcaccgt ctttcattgc
     2281 catacggaat tccggatgag cattcatcag gcgggcaaga atgtgaataa aggccggata
     2341 aaacttgtgc ttatttttct ttacggtctt taaaaaggcc gtaatatcca gctgaacggt
     2401 ctggttatag gtacattgag caactgactg aaatgcctca aaatgttctt tacgatgcca
     2461 ttgggatata tcaacggtgg tatatccagt gatttttttc tccattttag cttccttagc
     2521 tcctgaaaat ctcgataact caaaaaatac gcccggtagt gatcttattt cattatggtg
     2581 aaagttggaa cctcttacgt gccgatcaac gtctcatttt cgccaaaagt tggcccaggg
     2641 cttcccggta tcaacaggga caccaggatt tatttattct gcgaagtgat cttccgtcac
     2701 aggtatttat tcggcgcaaa gtgcgtcggg tgatgctgcc aacttagtcg actacaggtc
     2761 actaatacca tctaagtagt tgattcatag tgactggata tgttgtgttt tacagtatta
     2821 tgtagtctgt tttttatgca aaatctaatt taatatattg atatttatat cattttacgt
     2881 ttctcgttca gctttcttgt acaaagttgg cattataaga aagcattgct tatcaatttg
     2941 ttgcaacgaa caggtcacta tcagtcaaaa taaaatcatt atttgccatc cagctgatat
     3001 cccctatagt gagtcgtatt acatggtcat agctgtttcc tggcagctct ggcccgtgtc
     3061 tcaaaatctc tgatgttaca ttgcacaaga taaaaatata tcatcatgaa caataaaact
     3121 gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc
     3181 gaggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga
     3241 taatgtcggg caatcaggtg cgacaatcta tcgcttgtat gggaagcccg atgcgccaga
     3301 gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag
     3361 actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc
     3421 tgatgatgca tggttactca ccactgcgat ccccggaaaa acagcattcc aggtattaga
     3481 agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt
     3541 gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca
     3601 ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa
     3661 tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga
     3721 ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt
     3781 aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat
     3841 cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata
     3901 tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt
     3961 ctaatcagaa ttggttaatt ggttgtaaca ctggcagagc attacgctga cttgacggga
     4021 cggcgcaagc tcatgaccaa aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc
     4081 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     4141 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     4201 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     4261 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     4321 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     4381 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     4441 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     4501 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4561 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     4621 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     4681 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     4741 ctggcctttt gctcacatgt t
//
