LOCUS       sequence-237126-        2864 bp    DNA     circular SYN 12-MAY-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 2864)
  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 2864)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 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..2864
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    42..137
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(216..518)
                     /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="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDGSWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(318..337)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     promoter        complement(592..610)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(593..610)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    652..668
                     /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        complement(join(676..682,683..700,701..706))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    721..742
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    834..957
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1028..1045)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1028..1044)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1037..1059)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     terminator      1108..1194
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1286..1313
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1472..1489)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1643..2231)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1723..1742)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2321..2368
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2387..2761
                     /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"
     primer_bind     2829..2845
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(2850..2864,1..5)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(2850..2864,1..4)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 taggggatat cgcggccgcg ttaacgctac catggagctc caaataatga ttttattttg
       61 actgatagtg acctgttcgt tgcaacaaat tgataagcaa tgctttttta taatgccaac
      121 tttgtataat aaagttgaac cgcgagaccc acgctcaccc gccgcgctta atgcgccgct
      181 acagggcgcg tccattcgcc attcaggcct gacatttata ttccccagaa catcaggtta
      241 atggcgtttt tgatgtcatt ttcgcggtgg ctgagatcag ccacttcttc cccgataacg
      301 gagacaggca cactggccat atcggtggtc atcatgcgcc agcttccatc cccgatatgc
      361 accaccgggt aaagttcacg ggagacttta tctgacagca gacgtgcact ggccaggggg
      421 atcaccatcc gtcgccccgg cgtgtcaata atatcactct gtacatccac aaacagacga
      481 taacggctct ctcttttata ggtgtaaacc ttaaactgat gttcgagcgg tcgccagtgt
      541 gatgggtatc tacagactag acccattgag cttgatgcat agcttgagta ttctatagtg
      601 tcacctaaat agcttggcgt aatcatggtc atagctgttt cttgtgtgaa attgttatcc
      661 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
      721 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
      781 cctgtcgtgc cagctgcatt aatgaatcgg ccacttcaga ggtctctcga gttccacttt
      841 gtacaagaaa gttgaacgag aaacgtaaaa tgatataaat atcaatatat taaattagat
      901 tttgcataaa aaacagacta cataatactg taaaacacaa catatgcagt cactatgaac
      961 caactactta gatggtatta gtgacctgta gaattcgagc tctagagctg cagggcccga
     1021 gcttaagact ggccgtcgtt ttacaacgtc gtgactggga aaacatccat gctagcgtta
     1081 acgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc ggaagactgg
     1141 gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg
     1201 ggagcggatt tgaacgttgt gaagcaacgg cccggagggt ggcgggcagg acgcccgcca
     1261 taaactgcca ggcatcaaac taagcagaag gccatcctga cggatggcct ttttgcgttt
     1321 ctacaaactc ttcctggcta gcggtacgcg tattaattgc gttgcgctca ctgcccgctt
     1381 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     1441 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     1501 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     1561 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     1621 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     1681 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     1741 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     1801 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     1861 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     1921 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     1981 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     2041 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     2101 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     2161 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     2221 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgacg
     2281 cgtaactcac gttaagggat tttggtcatg atcagcacgt gttgacaatt aatcatcggc
     2341 atagtatatc ggcatagtat aatacgacaa ggtgaggaac taaaccatgg ccaagttgac
     2401 cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga
     2461 ccggctcggg ttctcccggg acttcgtgga ggacgacttc gccggtgtgg tccgggacga
     2521 cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg ccggacaaca ccctggcctg
     2581 ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa
     2641 cttccgggac gcctccgggc cggccatgac cgagatcggc gagcagccgt gggggcggga
     2701 gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg agcaggactg
     2761 atcatgatga tatattttta tcttgtgcaa tgtaacatca gagattttga gacacgggcc
     2821 agagctgcca ggaaacagct atgaccatgt aatacgactc acta
//
