LOCUS       sequence-203103-        4908 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pAH595-gRNA 2xplex entry cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4908)
  AUTHORS   Curtin SJ, Xiong Y, Michno JM, Campbell BW, Stec AO, Cermak T, 
            Starker C, Voytas DF, Eamens AL, Stupar RM
  TITLE     CRISPR/Cas9 and TALENs generate heritable mutations for genes 
            involved in small RNA processing of Glycine max and Medicago 
            truncatula.
  JOURNAL   Plant Biotechnol J. 2017 Oct 31. doi: 10.1111/pbi.12857.
  PUBMED    29087011
REFERENCE   2  (bases 1 to 4908)
  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 
            Biotechnol J. 2017 Oct 31. doi: 10.1111/pbi.12857."
COMMENT     Sequence Label: pSC10
FEATURES             Location/Qualifiers
     source          1..4908
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(312..638)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGAGLRTK
                     DRFV"
     primer_bind     503..519
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        529..547
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(595..615)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(595..613)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(634..650)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(647..669)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    658..674
                     /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(682..688,689..706,707..712))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    727..748
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     misc_RNA        789..864
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(876..900)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(1364..1669)
                     /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(1466..1485)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     misc_RNA        1995..2070
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(2082..2106)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2125..2224)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(2242..2260)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2265..2281)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2712..3503
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      3596..4184
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4085..4104
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4338..4355
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4514..4541)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4633..4719)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     4783..4799
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    join(4814..4908,1)
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 gccgagctcg cggccgcatg ttgttgttac cagaaagtaa ataaatgttc aatctctgat
       61 gttctcaagt aagtgagttt tattgggaat aatattaact tatgttcttc ttgcatttga
      121 tttctttgcc gctctcttct tctatcttaa atctgtgtat actatttcac tattgggctt
      181 tttattagtc tataatggga ctcaaaataa ggctttggcc cacatcaaaa agataagtca
      241 caaatcaaaa ctaaattcag agtcttttct cccacatcgg tcactgtact cattttgtgt
      301 ttgtttatat attacacgaa ccgatctttg gtacggagac cggcgccgct acagggcgcg
      361 tcccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
      421 ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca
      481 gggttttccc agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta atacgactca
      541 ctatagggcg aattgggtac cgggccgccc cttgaggtcc tccagctttt gttcccttta
      601 gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
      661 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
      721 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccaccg
      781 gtggtctcgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
      841 gaaaaagtgg caccgagtcg gtgctttttt tctagaccca gctttcttgt acaaagttgg
      901 cattaccatg gcttcgttga acaacggaaa ctcgacttgc cttccgcaca atacatcatt
      961 tcttcttagc tttttttctt cttcttcgtt catacagttt ttttttgttt atcagcttac
     1021 attttcttga accgtagctt tcgttttctt ctttttaact ttccattcgg agtttttgta
     1081 tcttgtttca tagtttgtcc caggattaga atgattaggc atcgaacctt caagaatttg
     1141 attgaataaa acatcttcat tcttaagata tgaagataat cttcaaaagg cccctgggaa
     1201 tctgaaagaa gagaagcagg cccatttata tgggaaagaa caatagtatt tcttatatag
     1261 gcccatttaa gttgaaaaca atcttcaaaa gtcccacatc gcttagataa gaaaacgaag
     1321 ctgagtttat atacagctag agtcgaagta gtgattggag acgttatatt ccccagaaca
     1381 tcaggttaat ggcgtttttg atgtcatttt cgcggtggct gagatcagcc acttcttccc
     1441 cgataacgga aaccggcaca ctggccatat cggtggtcat catgcgccag ctttcatccc
     1501 cgatatgcac caccgggtaa agttcacggg agactttatc tgacagcaga cgtgcactgg
     1561 ccagggggat caccatccgt cgcccgggcg tgtcaataat atcactctgt acatccacaa
     1621 acagacgata acggctctct cttttatagg tgtaaacctt aaactgcatt tcaccagccc
     1681 ctgttctcgt cagcaaaaga gccgttcatt tcaataaacc gggcgacctc agccatccct
     1741 tcctgatttt ccgctttcca gcgttcggca cgcagacgac gggcttcatt ctgcatggtt
     1801 gtgcttacca gaccggagat attgacatca tatatgcctt gagcaactga tagctgtcgc
     1861 tgtcaactgt cactgtaata cgctgcttca tagcatacct ccttttgaca tacttcgggt
     1921 atacatatca gtatatattc ttataccgca aaaatcagcg cgcaaatacg catactgtta
     1981 tctggcttcg tctcgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat
     2041 caacttgaaa aagtggcacc gagtcggtgc tttttttcta gacccagctt tcttgtacaa
     2101 agttggcatt actgcaggtc gacgacccag ctttcttgta caaagttggc attataaaaa
     2161 ataattgctc atcaatttgt tgcaacgaac aggtcactat cagtcaaaat aaaatcatta
     2221 tttgccatcc agctgatatc ccctatagtg agtcgtatta catggtcata gctgtttcct
     2281 ggcagctctg gcccgtgtct caaaatctct gatgttacat tgcacaagat aaaaatatat
     2341 catcatgcct cctctggacc agccaggaca gaaatgcctc gacttcgctg ctacccaagg
     2401 ttgccgggtg acgcacaccg tggaaacgga tgaaggcacg aacccagtgg acataagcct
     2461 gttcggttcg taagctgtaa tgcaagtagc gtatgcgctc acgcaactgg tccagaacct
     2521 tgaccgaacg cagcggtggt aacggcgcag tggcggtttt catggcttgt tatgactgtt
     2581 tttttggggt acagtctatg cctcgggcat ccaagcagca agcgcgttac gccgtgggtc
     2641 gatgtttgat gttatggagc agcaacgatg ttacgcagca gggcagtcgc cctaaaacaa
     2701 agttaaacat tatgagggaa gcggtgatcg ccgaagtatc gactcaacta tcagaggtag
     2761 ttggcgtcat cgagcgccat ctcgaaccga cgttgctggc cgtacatttg tacggctccg
     2821 cagtggatgg cggcctgaag ccacacagtg atattgattt gctggttacg gtgaccgtaa
     2881 ggcttgatga aacaacgcgg cgagctttga tcaacgacct tttggaaact tcggcttccc
     2941 ctggagagag cgagattctc cgcgctgtag aagtcaccat tgttgtgcac gacgacatca
     3001 ttccgtggcg ttatccagct aagcgcgaac tgcaatttgg agaatggcag cgcaatgaca
     3061 ttcttgcagg tatcttcgag ccagccacga tcgacattga tctggctatc ttgctgacaa
     3121 aagcaagaga acatagcgtt gccttggtag gtccagcggc ggaggaactc tttgatccgg
     3181 ttcctgaaca ggatctattt gaggcgctaa atgaaacctt aacgctatgg aactcgccgc
     3241 ccgactgggc tggcgatgag cgaaatgtag tgcttacgtt gtcccgcatt tggtacagcg
     3301 cagtaaccgg caaaatcgcg ccgaaggatg tcgctgccga ctgggcaatg gagcgcctgc
     3361 cggcccagta tcagcccgtc atacttgaag ctagacaggc ttatcttgga caagaagaag
     3421 atcgcttggc ctcgcgcgca gatcagttgg aagaatttgt ccactacgtg aaaggcgaga
     3481 tcaccaaggt agtcggcaaa taaccctcga gccacccatg accaaaatcc cttaacgtga
     3541 gttacgcgtc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3601 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     3661 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3721 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     3781 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     3841 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     3901 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     3961 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     4021 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     4081 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     4141 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     4201 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     4261 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     4321 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     4381 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     4441 actggaaagc gggcagtgag cgcaacgcaa ttaatacgcg taccgcgagc caggaagagt
     4501 ttgtagaaac gcaaaaaggc catccgtcag gatggccttc tgcttagttt gatgcctggc
     4561 agtttatggc gggcgtcctg cccgccaccc tccgggccgt tgcttcacaa cgttcaaatc
     4621 cgctcccggc ggatttgtcc tactcaggag agcgttcacc gacaaacaac agataaaacg
     4681 aaaggcccag tcttccgact gagcctttcg ttttatttga tgcctggcag ttccctactc
     4741 tcgcgttaac gcttgcatgg atgttttccc agtcacgacg ttgtaaaacg acggccagtc
     4801 ttaagctcgg gcccaataat gattttattt tgactgatag tgacctgttc gttgcaacaa
     4861 attgatgagc aatgcttttt tataatgcca actttgtata caaaagtt
//
