LOCUS       sequence-148627-        4374 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA shuttle vector; module 2.
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: pDGE7
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"
     promoter        551..653
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             654..1313
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(720..739)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1655..1960
                     /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     1839..1858
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     misc_RNA        1998..2073
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     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 tttggtctca cccttaagtt gaaaacaatc ttcaaaagtc ccacatcgct
      481 tagataagaa aacgaagctg agtttatata cagctagagt cgaagtagtg attgaagtct
      541 tctgagacgt tgatcggcac gtaagaggtt ccaactttca ccataatgaa ataagatcac
      601 taccgggcgt attttttgag ttatcgagat tttcaggagc taaggaagct aaaatggaga
      661 aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa gaacattttg
      721 aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg gatattacgg
      781 cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc
      841 ttgcccgcct gatgaatgct catccggaat tccgtatggc aatgaaagac ggtgagctgg
      901 tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt
      961 catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag
     1021 atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt
     1081 ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata
     1141 tggacaactt cttcgccccc gttttcacca tgggcaaata ttatacgcaa ggcgacaagg
     1201 tgctgatgcc gctggcgatt caggttcatc atgccgtctg tgatggcttc catgtcggca
     1261 gaatgcttaa tgaattacaa cagtactgcg atgagtggca gggcggggcg taatcgcgtg
     1321 gatccggctt actaaaagcc agataacagt atgcgtattt gcgcgctgat ttttgcggta
     1381 taagaatata tactgatatg tatacccgaa gtatgtcaaa aagaggtatg ctatgaagca
     1441 gcgtattaca gtgacagttg acagcgacag ctatcagttg ctcaaggcat atatgatgtc
     1501 aatatctccg gtctggtaag cacaaccatg cagaatgaag cccgtcgtct gcgtgccgaa
     1561 cgctggaaag cggaaaatca ggaagggatg gctgaggtcg cccggtttat tgaaatgaac
     1621 ggctcttttg ctgacgagaa caggggctgg tgaaatgcag tttaaggttt acacctataa
     1681 aagagagagc cgttatcgtc tgtttgtgga tgtacagagt gatattattg acacgcccgg
     1741 gcgacggatg gtgatccccc tggccagtgc acgtctgctg tcagataaag tctcccgtga
     1801 actttacccg gtggtgcata tcggggatga aagctggcgc atgatgacca ccgatatggc
     1861 cagtgtgccg gtttccgtta tcggggaaga agtggctgat ctcagccacc gcgaaaatga
     1921 catcaaaaac gccattaacc tgatgttctg gggaatataa atgtcaggct cccttataca
     1981 cagccagtcg aagacttgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt
     2041 tatcaacttg aaaaagtggc accgagtcgg tgcttttttt tcggttgaga 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
//
