LOCUS       sequence-148635-        4374 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA shuttle vector; module 8E.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4374)
  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 4374)
  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: pDGE15
FEATURES             Location/Qualifiers
     source          1..4374
                     /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"
     misc_RNA        complement(453..528)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(566..871)
                     /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(668..687)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1213..1872)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1787..1806
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1873..1975)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(2153..2169)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2153..2169)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2166..2188)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2177..2193
                     /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(2201..2207,2208..2225,2226..2231))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2246..2267
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2384..2401)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2555..3143)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2635..2654)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3314..4105,4106..4174))
                     /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     3937..3956
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4175..4279)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4347..4365
                     /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 taaggtctct aagcaaaaaa aagcaccgac tcggtgccac tttttcaagt
      481 tgataacgga ctagccttat tttaacttgc tatttctagc tctaaaacaa gtcttcgact
      541 ggctgtgtat aagggagcct gacatttata ttccccagaa catcaggtta atggcgtttt
      601 tgatgtcatt ttcgcggtgg ctgagatcag ccacttcttc cccgataacg gaaaccggca
      661 cactggccat atcggtggtc atcatgcgcc agctttcatc cccgatatgc accaccgggt
      721 aaagttcacg ggagacttta tctgacagca gacgtgcact ggccaggggg atcaccatcc
      781 gtcgcccggg cgtgtcaata atatcactct gtacatccac aaacagacga taacggctct
      841 ctcttttata ggtgtaaacc ttaaactgca tttcaccagc ccctgttctc gtcagcaaaa
      901 gagccgttca tttcaataaa ccgggcgacc tcagccatcc cttcctgatt ttccgctttc
      961 cagcgttcgg cacgcagacg acgggcttca ttctgcatgg ttgtgcttac cagaccggag
     1021 atattgacat catatatgcc ttgagcaact gatagctgtc gctgtcaact gtcactgtaa
     1081 tacgctgctt catagcatac ctctttttga catacttcgg gtatacatat cagtatatat
     1141 tcttataccg caaaaatcag cgcgcaaata cgcatactgt tatctggctt ttagtaagcc
     1201 ggatccacgc gattacgccc cgccctgcca ctcatcgcag tactgttgta attcattaag
     1261 cattctgccg acatggaagc catcacagac ggcatgatga acctgaatcg ccagcggcat
     1321 cagcaccttg tcgccttgcg tataatattt gcccatggtg aaaacggggg cgaagaagtt
     1381 gtccatattg gccacgttta aatcaaaact ggtgaaactc acccagggat tggctgagac
     1441 gaaaaacata ttctcaataa accctttagg gaaataggcc aggttttcac cgtaacacgc
     1501 cacatcttgc gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt cactccagag
     1561 cgatgaaaac gtttcagttt gctcatggaa aacggtgtaa caagggtgaa cactatccca
     1621 tatcaccagc tcaccgtctt tcattgccat acggaattcc ggatgagcat tcatcaggcg
     1681 ggcaagaatg tgaataaagg ccggataaaa cttgtgctta tttttcttta cggtctttaa
     1741 aaaggccgta atatccagct gaacggtctg gttataggta cattgagcaa ctgactgaaa
     1801 tgcctcaaaa tgttctttac gatgccattg ggatatatca acggtggtat atccagtgat
     1861 ttttttctcc attttagctt ccttagctcc tgaaaatctc gataactcaa aaaatacgcc
     1921 cggtagtgat cttatttcat tatggtgaaa gttggaacct cttacgtgcc gatcaacgtc
     1981 tcagaagact tcaatcacta cttcgactct agctgtatat aaactcagct tcgttttctt
     2041 atctaagcga tgtgggactt ttgaagattg ttttcaactt attcgtgaga ccaaaatcgg
     2101 atcccgggcc cgtcgactgc agaggcctgc atgcaagctt ggcgtaatca tggtcatagc
     2161 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     2221 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     2281 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     2341 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     2401 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     2461 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     2521 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     2581 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     2641 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     2701 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     2761 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     2821 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     2881 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     2941 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     3001 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     3061 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     3121 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     3181 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     3241 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     3301 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     3361 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     3421 taccatctgg ccccagtgct gcaatgatac cgcgagaacc acgctcaccg gctccagatt
     3481 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     3541 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     3601 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     3661 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     3721 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     3781 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     3841 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     3901 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     3961 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     4021 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     4081 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     4141 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     4201 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     4261 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     4321 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtc
//
