LOCUS       sequence-148632-        4497 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA shuttle vector; module 5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4497)
  AUTHORS   Ordon J, Gantner J, Kemna J, Schwalgun L, Reschke M, Streubel J, 
            Boch J, Stuttmann J
  TITLE     Generation of chromosomal deletions in dicotyledonous plants 
            employing a user-friendly genome editing toolkit.
  JOURNAL   Plant J. 2016 Aug 31. doi: 10.1111/tpj.13319.
  PUBMED    27579989
REFERENCE   2  (bases 1 to 4497)
  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: "Plant J. 
            2016 Aug 31. doi: 10.1111/tpj.13319."
COMMENT     Sequence Label: pDGE12
FEATURES             Location/Qualifiers
     source          1..4497
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        677..779
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             780..1439
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(846..865)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1781..2086
                     /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     1965..1984
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     misc_RNA        2124..2199
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2276..2292)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2276..2292)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2289..2311)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2300..2316
                     /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(2324..2330,2331..2348,2349..2354))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2369..2390
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2507..2524)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2678..3266)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2758..2777)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3437..4228,4229..4297))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4060..4079
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4298..4402)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4470..4488
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa
      421 tgcatctaga tggtctcaga ctgccagctt ctatgagtac tgagagaaga atcattctct
      481 tagcttttgg agttgagttg gttttaactg acccagctaa gggcatgaaa ggagctatcg
      541 caaaggcgga agagattttg gcgaaaggac taagttgaaa acaatcttca aaagtcccac
      601 atcgcttaga taagaaaacg aagctgagtt tatatacagc tagagtcgaa gtagtgattg
      661 aagtcttctg agacgttgat cggcacgtaa gaggttccaa ctttcaccat aatgaaataa
      721 gatcactacc gggcgtattt tttgagttat cgagattttc aggagctaag gaagctaaaa
      781 tggagaaaaa aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac
      841 attttgaggc atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata
      901 ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc
      961 acattcttgc ccgcctgatg aatgctcatc cggaattccg tatggcaatg aaagacggtg
     1021 agctggtgat atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa
     1081 cgttttcatc gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt
     1141 cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga
     1201 atatgttttt cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg
     1261 ccaatatgga caacttcttc gcccccgttt tcaccatggg caaatattat acgcaaggcg
     1321 acaaggtgct gatgccgctg gcgattcagg ttcatcatgc cgtctgtgat ggcttccatg
     1381 tcggcagaat gcttaatgaa ttacaacagt actgcgatga gtggcagggc ggggcgtaat
     1441 cgcgtggatc cggcttacta aaagccagat aacagtatgc gtatttgcgc gctgattttt
     1501 gcggtataag aatatatact gatatgtata cccgaagtat gtcaaaaaga ggtatgctat
     1561 gaagcagcgt attacagtga cagttgacag cgacagctat cagttgctca aggcatatat
     1621 gatgtcaata tctccggtct ggtaagcaca accatgcaga atgaagcccg tcgtctgcgt
     1681 gccgaacgct ggaaagcgga aaatcaggaa gggatggctg aggtcgcccg gtttattgaa
     1741 atgaacggct cttttgctga cgagaacagg ggctggtgaa atgcagttta aggtttacac
     1801 ctataaaaga gagagccgtt atcgtctgtt tgtggatgta cagagtgata ttattgacac
     1861 gcccgggcga cggatggtga tccccctggc cagtgcacgt ctgctgtcag ataaagtctc
     1921 ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga
     1981 tatggccagt gtgccggttt ccgttatcgg ggaagaagtg gctgatctca gccaccgcga
     2041 aaatgacatc aaaaacgcca ttaacctgat gttctgggga atataaatgt caggctccct
     2101 tatacacagc cagtcgaaga cttgttttag agctagaaat agcaagttaa aataaggcta
     2161 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttttcc gtgagaccat
     2221 cggatcccgg gcccgtcgac tgcagaggcc tgcatgcaag cttggcgtaa tcatggtcat
     2281 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     2341 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     2401 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     2461 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     2521 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     2581 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     2641 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     2701 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     2761 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     2821 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     2881 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     2941 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     3001 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     3061 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     3121 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     3181 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     3241 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     3301 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     3361 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     3421 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     3481 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     3541 gcttaccatc tggccccagt gctgcaatga taccgcgaga accacgctca ccggctccag
     3601 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     3661 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     3721 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     3781 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     3841 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     3901 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     3961 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     4021 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     4081 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     4141 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     4201 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     4261 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     4321 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     4381 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     4441 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtc
//
