LOCUS       sequence-220514-        2138 bp    DNA     circular SYN 14-APR-2025
DEFINITION  T7 promoter crRNA entry vector used for in vitro transcription of 
            AsCas12a guides.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2138)
  AUTHORS   Kleinstiver BP, Sousa AA, Walton RT, Tak YE, Hsu JY, Clement K, 
            Welch MM, Horng JE, Malagon-Lopez J, Scarfo I, Maus MV, Pinello L, 
            Aryee MJ, Joung JK
  TITLE     Engineered CRISPR-Cas12a variants with increased activities and 
            improved targeting ranges for gene, epigenetic and base editing.
  JOURNAL   Nat Biotechnol. 2019 Feb 11. pii: 10.1038/s41587-018-0011-0. doi: 
            10.1038/s41587-018-0011-0.
  PUBMED    30742127
REFERENCE   2  (bases 1 to 2138)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2019 Feb 11. pii: 10.1038/s41587-018-0011-0. doi: 
            10.1038/s41587-018-0011-0."
COMMENT     Sequence Label: pT7-G-AsCas12a crRNA-BsaI cassette (MSP3491)
FEATURES             Location/Qualifiers
     source          1..2138
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      36..623
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     524..543
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(635..662)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(686..1501)
                     /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     1409..1428
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1502..1604)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(1605..1636)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     primer_bind     1684..1700
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1684..1700
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2117..2134)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2117..2133)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 tgggcctttc ttcggtagaa aatcaaagga tcttcttgag atcctttttt tctgcgcgta
       61 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
      121 gagctaccaa ctctttttcc gaggtaactg gcttcagcag agcgcagata ccaaatactg
      181 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
      241 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
      301 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
      361 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
      421 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
      481 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
      541 tttatagtcc tgtcgggttt cgccacctct gacttgagca tcgatttttg tgatgctcgt
      601 caggggggcg gagcctatgg aaaaacgcca gcaacgcaga aaggcccacc cgaaggtgag
      661 ccaggtgatt acatttaggt cctcattaga aaaactcatc gagcatcaag tgaaactgca
      721 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag
      781 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc
      841 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa
      901 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaagagt ttatgcattt
      961 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcaccaa
     1021 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgccgttaa
     1081 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa
     1141 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttccctggga
     1201 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa
     1261 gaggcataaa ttccgtcagc cagtttagcc tgaccatctc atctgtaaca tcattggcaa
     1321 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat
     1381 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag
     1441 catccatgtt ggaatttaat cgcggcttca agcaagacgt ttcccgttga atatggctca
     1501 ttttagcttc cttagctcct gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc
     1561 ttatttcatt atggtgaaag ttggaacctc ttacgtgccg atcaagtcaa aagcctccgg
     1621 tcggaggctt ttgactttct gctatggagg tcaggtatga tttaaatggt cagtattgag
     1681 cctcaggaaa cagctatgac atcaagctga ctagataatc tagctgatcg tggaccgatc
     1741 atacgtataa tgccgtaaga tcacgggtcg cagcacagct cgcggtccag tagtgatcga
     1801 cactgctcga tccgctcgca ccgctagcta atacgactca ctatagtaat ttctactctt
     1861 gtagatggag acccatgcca tagcgttgtt cggaatatga atttttgaac agattcacca
     1921 acacctagtg gtctccttta aaaagcttgg atcgacgaga gcagcgcgac tggatctgtc
     1981 gcccgtctca aacgcaaccc tccggcggtc gcatatcatt caggacgagc ctcagactcc
     2041 agcgtaactg gactgcaatc aactcactgg ctcaccttcc ggtccacgat cagctagaat
     2101 caagctgact agataaactg gccgtcgttt tacacggg
//
