LOCUS       sequence-148628-        4375 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA shuttle vector; module 2E.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4375)
  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 4375)
  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: pDGE8
FEATURES             Location/Qualifiers
     source          1..4375
                     /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        552..654
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             655..1314
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(721..740)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1656..1961
                     /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     1840..1859
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     misc_RNA        1999..2074
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2154..2170)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2154..2170)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2167..2189)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2178..2194
                     /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(2202..2208,2209..2226,2227..2232))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2247..2268
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2385..2402)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2556..3144)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2636..2655)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3315..4106,4107..4175))
                     /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     3938..3957
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4176..4280)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4348..4366
                     /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 ttttggtctc acccttaagt tgaaaacaat cttcaaaagt cccacatcgc
      481 ttagataaga aaacgaagct gagtttatat acagctagag tcgaagtagt gattgaagtc
      541 ttctgagacg ttgatcggca cgtaagaggt tccaactttc accataatga aataagatca
      601 ctaccgggcg tattttttga gttatcgaga ttttcaggag ctaaggaagc taaaatggag
      661 aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt
      721 gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg
      781 gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt
      841 cttgcccgcc tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga cggtgagctg
      901 gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt
      961 tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa
     1021 gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg
     1081 tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat
     1141 atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag
     1201 gtgctgatgc cgctggcgat tcaggttcat catgccgtct gtgatggctt ccatgtcggc
     1261 agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc gtaatcgcgt
     1321 ggatccggct tactaaaagc cagataacag tatgcgtatt tgcgcgctga tttttgcggt
     1381 ataagaatat atactgatat gtatacccga agtatgtcaa aaagaggtat gctatgaagc
     1441 agcgtattac agtgacagtt gacagcgaca gctatcagtt gctcaaggca tatatgatgt
     1501 caatatctcc ggtctggtaa gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga
     1561 acgctggaaa gcggaaaatc aggaagggat ggctgaggtc gcccggttta ttgaaatgaa
     1621 cggctctttt gctgacgaga acaggggctg gtgaaatgca gtttaaggtt tacacctata
     1681 aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgcccg
     1741 ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg
     1801 aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg
     1861 ccagtgtgcc ggtttccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg
     1921 acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc tcccttatac
     1981 acagccagtc gaagacttgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
     2041 ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt ttgcttagag acctttatcg
     2101 gatcccgggc ccgtcgactg cagaggcctg catgcaagct tggcgtaatc atggtcatag
     2161 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     2221 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     2281 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     2341 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     2401 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     2461 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     2521 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     2581 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     2641 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     2701 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     2761 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     2821 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     2881 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     2941 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     3001 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     3061 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     3121 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     3181 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     3241 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     3301 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     3361 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     3421 ttaccatctg gccccagtgc tgcaatgata ccgcgagaac cacgctcacc ggctccagat
     3481 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     3541 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     3601 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     3661 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     3721 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     3781 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     3841 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     3901 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     3961 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     4021 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     4081 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     4141 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     4201 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     4261 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     4321 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtc
//
