LOCUS       sequence-333508-        2091 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  T7 promoter expression plasmid for in vitro transcription of 
            AsCas12a crRNA with spacer #3.
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-site3 (RTW549)
FEATURES             Location/Qualifiers
     source          1..2091
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     25..41
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     25..41
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(411..428)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(411..427)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      468..1055
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     956..975
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1067..1094)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(1118..1933)
                     /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     1841..1860
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1934..2036)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(2037..2068)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
ORIGIN
        1 atttaaatgg tcagtattga gcctcaggaa acagctatga catcaagctg actagataat
       61 ctagctgatc gtggaccgat catacgtata atgccgtaag atcacgggtc gcagcacagc
      121 tcgcggtcca gtagtgatcg acactgctcg atccgctcgc accgctagct aatacgactc
      181 actatagtaa tttctactct tgtagatctg atggtccatg tctgttactc tttaaaaagc
      241 ttggatcgac gagagcagcg cgactggatc tgtcgcccgt ctcaaacgca accctccggc
      301 ggtcgcatat cattcaggac gagcctcaga ctccagcgta actggactgc aatcaactca
      361 ctggctcacc ttccggtcca cgatcagcta gaatcaagct gactagataa actggccgtc
      421 gttttacacg ggtgggcctt tcttcggtag aaaatcaaag gatcttcttg agatcctttt
      481 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
      541 ttgccggatc aagagctacc aactcttttt ccgaggtaac tggcttcagc agagcgcaga
      601 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
      661 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
      721 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
      781 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
      841 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
      901 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
      961 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag catcgatttt
     1021 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgca gaaaggccca
     1081 cccgaaggtg agccaggtga ttacatttag gtcctcatta gaaaaactca tcgagcatca
     1141 agtgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     1201 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     1261 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     1321 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaga
     1381 gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     1441 cactcgcacc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
     1501 gatcgccgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     1561 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     1621 ttttccctgg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     1681 tgatggtcgg aagaggcata aattccgtca gccagtttag cctgaccatc tcatctgtaa
     1741 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
     1801 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     1861 catataaatc agcatccatg ttggaattta atcgcggctt caagcaagac gtttcccgtt
     1921 gaatatggct cattttagct tccttagctc ctgaaaatct cgataactca aaaaatacgc
     1981 ccggtagtga tcttatttca ttatggtgaa agttggaacc tcttacgtgc cgatcaagtc
     2041 aaaagcctcc ggtcggaggc ttttgacttt ctgctatgga ggtcaggtat g
//
