LOCUS       sequence-198619-        4909 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 4909)
  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 4909)
  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..4909
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(310..636)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGAGLRTK
                     DRFV"
     primer_bind     501..517
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        527..545
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(593..613)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(593..611)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(632..648)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(645..667)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    656..672
                     /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(680..686,687..704,705..710))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    725..746
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     misc_RNA        787..862
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(874..898)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(1362..1667)
                     /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(1464..1483)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     misc_RNA        1993..2068
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(2080..2104)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2123..2222)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(2240..2258)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2263..2279)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2710..3501
                     /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      3594..4182
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4083..4102
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4336..4353
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4512..4539)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4631..4717)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     4781..4797
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4812..4907
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 gagctcgcgg ccgcgatgtt gttgttacca gaaagtaaat aaatgttcaa tctctgatgt
       61 tctcaagtaa gtgagtttta ttgggaataa tattaactta tgttcttctt gcatttgatt
      121 tctttgccgc tctcttcttc tatcttaaat ctgtgtatac tatttcacta ttgggctttt
      181 tattagtcta taatgggact caaaataagg ctttggccca catcaaaaag ataagtcaca
      241 aatcaaaact aaattcagag tcttttctcc cacatcggtc actgtactca ttttgtgttt
      301 gtttatatat tacacgaacc gatctttggt acggagaccg gcgccgctac agggcgcgtc
      361 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
      421 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
      481 gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat acgactcact
      541 atagggcgaa ttgggtaccg ggccgcccct tgaggtcctc cagcttttgt tccctttagt
      601 gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
      661 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
      721 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccaccggt
      781 ggtctcgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
      841 aaaagtggca ccgagtcggt gctttttttc tagacccagc tttcttgtac aaagttggca
      901 ttaccatggc ttcgttgaac aacggaaact cgacttgcct tccgcacaat acatcatttc
      961 ttcttagctt tttttcttct tcttcgttca tacagttttt ttttgtttat cagcttacat
     1021 tttcttgaac cgtagctttc gttttcttct ttttaacttt ccattcggag tttttgtatc
     1081 ttgtttcata gtttgtccca ggattagaat gattaggcat cgaaccttca agaatttgat
     1141 tgaataaaac atcttcattc ttaagatatg aagataatct tcaaaaggcc cctgggaatc
     1201 tgaaagaaga gaagcaggcc catttatatg ggaaagaaca atagtatttc ttatataggc
     1261 ccatttaagt tgaaaacaat cttcaaaagt cccacatcgc ttagataaga aaacgaagct
     1321 gagtttatat acagctagag tcgaagtagt gattggagac gttatattcc ccagaacatc
     1381 aggttaatgg cgtttttgat gtcattttcg cggtggctga gatcagccac ttcttccccg
     1441 ataacggaaa ccggcacact ggccatatcg gtggtcatca tgcgccagct ttcatccccg
     1501 atatgcacca ccgggtaaag ttcacgggag actttatctg acagcagacg tgcactggcc
     1561 agggggatca ccatccgtcg cccgggcgtg tcaataatat cactctgtac atccacaaac
     1621 agacgataac ggctctctct tttataggtg taaaccttaa actgcatttc accagcccct
     1681 gttctcgtca gcaaaagagc cgttcatttc aataaaccgg gcgacctcag ccatcccttc
     1741 ctgattttcc gctttccagc gttcggcacg cagacgacgg gcttcattct gcatggttgt
     1801 gcttaccaga ccggagatat tgacatcata tatgccttga gcaactgata gctgtcgctg
     1861 tcaactgtca ctgtaatacg ctgcttcata gcatacctcc ttttgacata cttcgggtat
     1921 acatatcagt atatattctt ataccgcaaa aatcagcgcg caaatacgca tactgttatc
     1981 tggcttcgtc tcgttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca
     2041 acttgaaaaa gtggcaccga gtcggtgctt tttttctaga cccagctttc ttgtacaaag
     2101 ttggcattac tgcaggtcga cgacccagct ttcttgtaca aagttggcat tataaaaaat
     2161 aattgctcat caatttgttg caacgaacag gtcactatca gtcaaaataa aatcattatt
     2221 tgccatccag ctgatatccc ctatagtgag tcgtattaca tggtcatagc tgtttcctgg
     2281 cagctctggc ccgtgtctca aaatctctga tgttacattg cacaagataa aaatatatca
     2341 tcatgcctcc tctggaccag ccaggacaga aatgcctcga cttcgctgct acccaaggtt
     2401 gccgggtgac gcacaccgtg gaaacggatg aaggcacgaa cccagtggac ataagcctgt
     2461 tcggttcgta agctgtaatg caagtagcgt atgcgctcac gcaactggtc cagaaccttg
     2521 accgaacgca gcggtggtaa cggcgcagtg gcggttttca tggcttgtta tgactgtttt
     2581 tttggggtac agtctatgcc tcgggcatcc aagcagcaag cgcgttacgc cgtgggtcga
     2641 tgtttgatgt tatggagcag caacgatgtt acgcagcagg gcagtcgccc taaaacaaag
     2701 ttaaacatca tgagggaagc ggtgatcgcc gaagtatcga ctcaactatc agaggtagtt
     2761 ggcgtcatcg agcgccatct cgaaccgacg ttgctggccg tacatttgta cggctccgca
     2821 gtggatggcg gcctgaagcc acacagtgat attgatttgc tggttacggt gaccgtaagg
     2881 cttgatgaaa caacgcggcg agctttgatc aacgaccttt tggaaacttc ggcttcccct
     2941 ggagagagcg agattctccg cgctgtagaa gtcaccattg ttgtgcacga cgacatcatt
     3001 ccgtggcgtt atccagctaa gcgcgaactg caatttggag aatggcagcg caatgacatt
     3061 cttgcaggta tcttcgagcc agccacgatc gacattgatc tggctatctt gctgacaaaa
     3121 gcaagagaac atagcgttgc cttggtaggt ccagcggcgg aggaactctt tgatccggtt
     3181 cctgaacagg atctatttga ggcgctaaat gaaaccttaa cgctatggaa ctcgccgccc
     3241 gactgggctg gcgatgagcg aaatgtagtg cttacgttgt cccgcatttg gtacagcgca
     3301 gtaaccggca aaatcgcgcc gaaggatgtc gctgccgact gggcaatgga gcgcctgccg
     3361 gcccagtatc agcccgtcat acttgaagct agacaggctt atcttggaca agaagaagat
     3421 cgcttggcct cgcgcgcaga tcagttggaa gaatttgtcc actacgtgaa aggcgagatc
     3481 accaaggtag tcggcaaata accctcgagc cacccatgac caaaatccct taacgtgagt
     3541 tacgcgtcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     3601 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     3661 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     3721 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     3781 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     3841 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     3901 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     3961 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     4021 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     4081 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     4141 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     4201 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     4261 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     4321 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
     4381 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     4441 tggaaagcgg gcagtgagcg caacgcaatt aatacgcgta ccgcgagcca ggaagagttt
     4501 gtagaaacgc aaaaaggcca tccgtcagga tggccttctg cttagtttga tgcctggcag
     4561 tttatggcgg gcgtcctgcc cgccaccctc cgggccgttg cttcacaacg ttcaaatccg
     4621 ctcccggcgg atttgtccta ctcaggagag cgttcaccga caaacaacag ataaaacgaa
     4681 aggcccagtc ttccgactga gcctttcgtt ttatttgatg cctggcagtt ccctactctc
     4741 gcgttaacgc ttgcatggat gttttcccag tcacgacgtt gtaaaacgac ggccagtctt
     4801 aagctcgggc ccaataatga ttttattttg actgatagtg acctgttcgt tgcaacaaat
     4861 tgatgagcaa tgctttttta taatgccaac tttgtataca aaagttgcc
//
