LOCUS       sequence-333510-        2091 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  T7 promoter expression plasmid for in vitro transcription of 
            AsCas12a crRNA with spacer #4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2091)
  AUTHORS   Walton RT, Hsu JY, Joung JK, Kleinstiver BP
  TITLE     Scalable characterization of the PAM requirements of CRISPR-Cas 
            enzymes using HT-PAMDA.
  JOURNAL   Nat Protoc. 2021 Feb 5. pii: 10.1038/s41596-020-00465-2. doi: 
            10.1038/s41596-020-00465-2.
  PUBMED    33547443
REFERENCE   2  (bases 1 to 2091)
  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: "Nat Protoc.
            2021 Feb 5. pii: 10.1038/s41596-020-00465-2. doi: 
            10.1038/s41596-020-00465-2."
COMMENT     Sequence Label: pT7-AsCas12a_crRNA-site4 (MSP3511)
FEATURES             Location/Qualifiers
     source          1..2091
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(345..362)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(345..361)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      402..989
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     890..909
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1001..1028)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(1052..1867)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCLKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1775..1794
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1868..1970)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(1971..2002)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     primer_bind     2050..2066
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2050..2066
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gatcgtggac cgatcatacg tataatgccg taagatcacg ggtcgcagca cagctcgcgg
       61 tccagtagtg atcgacactg ctcgatccgc tcgcaccgct agctaatacg actcactata
      121 gtaatttcta ctcttgtaga tggaatccct tctgcagcac ctggtttaaa aagcttggat
      181 cgacgagagc agcgcgactg gatctgtcgc ccgtctcaaa cgcaaccctc cggcggtcgc
      241 atatcattca ggacgagcct cagactccag cgtaactgga ctgcaatcaa ctcactggct
      301 caccttccgg tccacgatca gctagaatca agctgactag ataaactggc cgtcgtttta
      361 cacgggtggg cctttcttcg gtagaaaatc aaaggatctt cttgagatcc tttttttctg
      421 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
      481 gatcaagagc taccaactct ttttccgagg taactggctt cagcagagcg cagataccaa
      541 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
      601 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
      661 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
      721 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
      781 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
      841 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
      901 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcatcga tttttgtgat
      961 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcagaaagg cccacccgaa
     1021 ggtgagccag gtgattacat ttaggtcctc attagaaaaa ctcatcgagc atcaagtgaa
     1081 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     1141 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     1201 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     1261 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aagagtttat
     1321 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     1381 caccaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     1441 cgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     1501 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     1561 ctgggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     1621 tcggaagagg cataaattcc gtcagccagt ttagcctgac catctcatct gtaacatcat
     1681 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     1741 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     1801 aatcagcatc catgttggaa tttaatcgcg gcttcaagca agacgtttcc cgttgaatat
     1861 ggctcatttt agcttcctta gctcctgaaa atctcgataa ctcaaaaaat acgcccggta
     1921 gtgatcttat ttcattatgg tgaaagttgg aacctcttac gtgccgatca agtcaaaagc
     1981 ctccggtcgg aggcttttga ctttctgcta tggaggtcag gtatgattta aatggtcagt
     2041 attgagcctc aggaaacagc tatgacatca agctgactag ataatctagc t
//
