LOCUS       sequence-148630-        4369 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA shuttle vector; module 4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4369)
  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 4369)
  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: pDGE10
FEATURES             Location/Qualifiers
     source          1..4369
                     /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        549..651
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             652..1311
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(718..737)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1653..1958
                     /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     1837..1856
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     misc_RNA        1996..2071
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2148..2164)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2148..2164)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2161..2183)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2172..2188
                     /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(2196..2202,2203..2220,2221..2226))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2241..2262
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2379..2396)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2550..3138)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2630..2649)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3309..4100,4101..4169))
                     /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     3932..3951
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4170..4274)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4342..4360
                     /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 tggtctcaat ggtaagttga aaacaatctt caaaagtccc acatcgctta
      481 gataagaaaa cgaagctgag tttatataca gctagagtcg aagtagtgat tgaagtcttc
      541 tgagacgttg atcggcacgt aagaggttcc aactttcacc ataatgaaat aagatcacta
      601 ccgggcgtat tttttgagtt atcgagattt tcaggagcta aggaagctaa aatggagaaa
      661 aaaatcactg gatataccac cgttgatata tcccaatggc atcgtaaaga acattttgag
      721 gcatttcagt cagttgctca atgtacctat aaccagaccg ttcagctgga tattacggcc
      781 tttttaaaga ccgtaaagaa aaataagcac aagttttatc cggcctttat tcacattctt
      841 gcccgcctga tgaatgctca tccggaattc cgtatggcaa tgaaagacgg tgagctggtg
      901 atatgggata gtgttcaccc ttgttacacc gttttccatg agcaaactga aacgttttca
      961 tcgctctgga gtgaatacca cgacgatttc cggcagtttc tacacatata ttcgcaagat
     1021 gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag ggtttattga gaatatgttt
     1081 ttcgtctcag ccaatccctg ggtgagtttc accagttttg atttaaacgt ggccaatatg
     1141 gacaacttct tcgcccccgt tttcaccatg ggcaaatatt atacgcaagg cgacaaggtg
     1201 ctgatgccgc tggcgattca ggttcatcat gccgtctgtg atggcttcca tgtcggcaga
     1261 atgcttaatg aattacaaca gtactgcgat gagtggcagg gcggggcgta atcgcgtgga
     1321 tccggcttac taaaagccag ataacagtat gcgtatttgc gcgctgattt ttgcggtata
     1381 agaatatata ctgatatgta tacccgaagt atgtcaaaaa gaggtatgct atgaagcagc
     1441 gtattacagt gacagttgac agcgacagct atcagttgct caaggcatat atgatgtcaa
     1501 tatctccggt ctggtaagca caaccatgca gaatgaagcc cgtcgtctgc gtgccgaacg
     1561 ctggaaagcg gaaaatcagg aagggatggc tgaggtcgcc cggtttattg aaatgaacgg
     1621 ctcttttgct gacgagaaca ggggctggtg aaatgcagtt taaggtttac acctataaaa
     1681 gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac acgcccgggc
     1741 gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc tcccgtgaac
     1801 tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc gatatggcca
     1861 gtgtgccggt ttccgttatc ggggaagaag tggctgatct cagccaccgc gaaaatgaca
     1921 tcaaaaacgc cattaacctg atgttctggg gaatataaat gtcaggctcc cttatacaca
     1981 gccagtcgaa gacttgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta
     2041 tcaacttgaa aaagtggcac cgagtcggtg cttttttttg acttgagacc atcggatccc
     2101 gggcccgtcg actgcagagg cctgcatgca agcttggcgt aatcatggtc atagctgttt
     2161 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     2221 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     2281 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     2341 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     2401 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     2461 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     2521 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     2581 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     2641 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     2701 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     2761 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     2821 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     2881 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     2941 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     3001 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     3061 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     3121 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     3181 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     3241 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     3301 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     3361 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     3421 tctggcccca gtgctgcaat gataccgcga gaaccacgct caccggctcc agatttatca
     3481 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     3541 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     3601 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     3661 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     3721 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     3781 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     3841 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     3901 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     3961 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     4021 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     4081 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     4141 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     4201 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     4261 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     4321 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtc
//
