LOCUS       sequence-311736-        3246 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  15 designed non-target sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3246)
  AUTHORS   Moreb EA, Hutmacher M, Lynch MD
  TITLE     CRISPR-Cas ""Non-Target"" Sites Inhibit On-Target Cutting Rates.
  JOURNAL   CRISPR J. 2020 Dec;3(6):550-561. doi: 10.1089/crispr.2020.0065.
  PUBMED    33346713
REFERENCE   2  (bases 1 to 3246)
  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"; doi: 
            "10.1089/crispr.2020"; journalName: "CRISPR J"; date: "2020-12"; 
            volume: "3"; issue: "6"; pages: "550-561"
COMMENT     Sequence Label: pNT-15
FEATURES             Location/Qualifiers
     source          1..3246
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      65..96
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     promoter        97..199
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             200..1015
                     /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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(273..292)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      1036..1063
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      complement(1075..1662)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1155..1174)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1684..1713)
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     CDS             complement(3185..3196)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
ORIGIN
        1 gacgaattct ctagatatcg ctcaatactg accatttaaa tcatacctga cctccatagc
       61 agaaagtcaa aagcctccga ccggaggctt ttgacttgat cggcacgtaa gaggttccaa
      121 ctttcaccat aatgaaataa gatcactacc gggcgtattt tttgagttat cgagattttc
      181 aggagctaag gaagctaaaa tgagccatat tcaacgggaa acgtcttgct cgaggccgcg
      241 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg
      301 gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag agttgtttct
      361 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca ggctaaactg
      421 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc
      481 atggttactc accactgcga tcccagggaa aacagcattc caggtattag aagaatatcc
      541 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat
      601 tcctgtttgt aattgtcctt ttaacggcga tcgcgtattt cgtctcgctc aggcgcaatc
      661 acgaatgaat aacggtttgg ttggtgcgag tgattttgat gacgagcgta atggctggcc
      721 tgttgaacaa gtctggaaag aaatgcataa gcttttgcca ttctcaccgg attcagtcgt
      781 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
      841 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
      901 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
      961 taatcctgat atgaataaat tgcagtttca cttgatgctc gatgagtttt tctgagggcc
     1021 caaatgtaat cacctggctc accttcgggt gggcctttct gcgttgctgg cgtttttcca
     1081 taggctccgc ccccctgacg agcatcacaa aaatcgatgc tcaagtcaga ggtggcgaaa
     1141 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     1201 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     1261 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     1321 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     1381 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     1441 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     1501 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttacctcgga
     1561 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     1621 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgattttc
     1681 taccgaagaa aggcccaccc gtgaaggtga gccagtgagt tgattgcagt ccagttacgc
     1741 tggagtctga ggctcgtcct gaatgatatc aagcttgaat tcgttttctg tggcacgact
     1801 tctctggact gttcttatca actccgaggg tagactcaat ctgaatactt ttcgttactg
     1861 gaccctggcc gtggggtaaa acggttggcg aactacgata cggaagtaac agtctcccta
     1921 ttattctgat tggtagcagt taactatcgg tctaaccctg gccgtgtggg tgggcgtctt
     1981 tgagaactgt tgactacacg cttatctgcc gaaatagaat actgagtgcc tttataaacg
     2041 gttgactacc ctggccgtga ggtaagtaga tttcttcgga ggaataatcg ctgagagtca
     2101 ctgaaatcaa tagtatcaca cctgtttacg actttactga accctggccg tgcgggaaac
     2161 ctgttatcta ccgattcaga agttcctatt gagttactgg gagactatca tagttgccag
     2221 agtgaatccc tataccctgg ccgtgcggga gaactttcag ccactatcac ggtagaagaa
     2281 tccttttcgg agtattttat tggtattcac gcaattgcca acagttaccc tggccgtgtg
     2341 gttgattcgg actgcgatag taaactgtag ataataagac cgctctgaca atacctcagg
     2401 ttttacgact atccagttaa ccctggccgt gaggctcagt aaacgaaata atacgataag
     2461 tagttcccaa tctctggttg tctcggtaga ctcagactaa ttgttacgga caaccctggc
     2521 cgtggggtac gaagactgat tgaggacgca actacaggtt ctattttact tattacgcca
     2581 gataggtaaa ccagaaatcc tcgttaccct ggccgtgcgg actctgaaaa tccgtgttga
     2641 ataaaggtgt agtagccgtt acgcaatacc aatctgaata cacttataat accgtgtcac
     2701 cctggccgtg aggaaacact ggattgactt ttcaggaaga ttcgcaactc gtagaagcgt
     2761 gattactagt tacaacgacg aaagcgtatt taccctggcc gtgtggtaag atagtaataa
     2821 ccaaccctgt ttccgctgta gtgtaagtgc ctttctgaag aattcaatcg taatctattg
     2881 gcgaaccctg gccgtgggga gacaatagga tttcgctggt ttcactctcg gaataaagtt
     2941 ctcagtagac tcaaagacga ctttgattat ctgagacacc ctggccgtgt ggatcccacg
     3001 ctgtattatc agattctatc ctattcgtag ttggaaaagt aagaggcagt gagtaacaag
     3061 tgcttccaat accctggccg tgaggttatc aactttcagg taatctctat tgtaagacca
     3121 cgagggacag gcgtattcta tagtgacaac ttatcagcac gataccctgg ccgtgcggta
     3181 actttctacc ctctattgaa ggataaggtg ttgattggac gattacagtc aggaaccagc
     3241 ctcagt
//
