LOCUS       sequence-470418-        2930 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry clone containing ccdB negative selection marker. Used to clone
            any DNA fragments of interest; goldengate and gibson assembly 
            cloning compatible. For use in plants and compatible with the 
            MultiSite Gateway system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2930)
  AUTHORS   Wang X, Ye L, Lyu M, Ursache R, Loytynoja A, Mahonen AP
  TITLE     An inducible genome editing system for plants.
  JOURNAL   Nat Plants. 2020 Jun 29. pii: 10.1038/s41477-020-0695-2. doi: 
            10.1038/s41477-020-0695-2.
  PUBMED    32601420
REFERENCE   2  (bases 1 to 2930)
  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: "Nat Plants.
            2020 Jun 29. pii: 10.1038/s41477-020-0695-2. doi: 
            10.1038/s41477-020-0695-2."
COMMENT     Sequence Label: p2R3z-BASI-ccdB-BSAI
FEATURES             Location/Qualifiers
     source          1..2930
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(919..946)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(1038..1124)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     1173..1195
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1187..1204
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1188..1204
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(1275..1398)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    1490..1511
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1526..1531,1532..1549,1550..1556)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1564..1580
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        1622..1640
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1622..1639
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     1895..1914
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2161..2256)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2293..2312)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2294..2312)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2317..2333)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(2401..2775)
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     promoter        complement(2794..2841)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat acgcgtaccg ctagccagga
      901 agagtttgta gaaacgcaaa aaggccatcc gtcaggatgg ccttctgctt agtttgatgc
      961 ctggcagttt atggcgggcg tcctgcccgc caccctccgg gccgttgctt cacaacgttc
     1021 aaatccgctc ccggcggatt tgtcctactc aggagagcgt tcaccgacaa acaacagata
     1081 aaacgaaagg cccagtcttc cgactgagcc tttcgtttta tttgatgcct ggcagttccc
     1141 tactctcgcg ttaacgctag catggatgtt ttcccagtca cgacgttgta aaacgacggc
     1201 cagtcttaag ctcgggccct gcagctctag agctcgaatt ctacaggtca ctaataccat
     1261 ctaagtagtt ggttcatagt gactgcatat gttgtgtttt acagtattat gtagtctgtt
     1321 ttttatgcaa aatctaattt aatatattga tatttatatc attttacgtt tctcgttcaa
     1381 ctttcttgta caaagtggaa ctcgagagac ctctgaagtg gccgattcat taatgcagct
     1441 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     1501 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     1561 gaattgtgag cggataacaa tttcacacaa gaaacagcta tgaccatgat tacgccaagc
     1621 tatttaggtg acactataga atactcaagc tatgcatcaa gctcaatggg tctagtctgt
     1681 agatacccat cacactggcg accgctcgaa catcagttta aggtttacac ctataaaaga
     1741 gagagccgtt atcgtctgtt tgtggatgta cagagtgata ttattgacac gccggggcga
     1801 cggatggtga tccccctggc cagtgcacgt ctgctgtcag ataaagtctc ccgtgaactt
     1861 tacccggtgg tgcatatcgg ggatggaagc tggcgcatga tgaccaccga tatggccagt
     1921 gtgcctgtct ccgttatcgg ggaagaagtg gctgagatca gccacttctt ccccgatatg
     1981 gccagtgtgc ctgtctccgt tatcggggaa gaagtggctg atctcagcca ccgcgaaaat
     2041 gacatcaaaa acgccattaa cctgatgttc tggggaatat aaatgtcagg cctgaatggc
     2101 gaatggacgc gccctgtagc ggcgcattaa gcgcggcggg tgagcgtggg tctcgcggtt
     2161 caactttatt atacaaagtt ggcattataa aaaagcattg cttatcaatt tgttgcaacg
     2221 aacaggtcac tatcagtcaa aataaaatca ttatttggag ctccatggta gcgttaacgc
     2281 ggccgcgata tcccctatag tgagtcgtat tacatggtca tagctgtttc ctggcagctc
     2341 tggcccgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatga
     2401 tcagtcctgc tcctcggcca cgaagtgcac gcagttgccg gccgggtcgc gcagggcgaa
     2461 ctcccgcccc cacggctgct cgccgatctc ggtcatggcc ggcccggagg cgtcccggaa
     2521 gttcgtggac acgacctccg accactcggc gtacagctcg tccaggccgc gcacccacac
     2581 ccaggccagg gtgttgtccg gcaccacctg gtcctggacc gcgctgatga acagggtcac
     2641 gtcgtcccgg accacaccgg cgaagtcgtc ctccacgaag tcccgggaga acccgagccg
     2701 gtcggtccag aactcgaccg ctccggcgac gtcgcgcgcg gtgagcaccg gaacggcact
     2761 ggtcaacttg gccatggttt agttcctcac cttgtcgtat tatactatgc cgatatacta
     2821 tgccgatgat taattgtcaa cacgtgctga tcatgaccaa aatcccttaa cgtgagttac
     2881 gcgtcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
//
