LOCUS       sequence-246076-        3060 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning plasmid for a self-targeting gRNA library.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3060)
  AUTHORS   Talas A, Huszar K, Kulcsar PI, Varga JK, Varga E, Toth E, Welker Z, 
            Erdos G, Pach PF, Welker A, Gyorgypal Z, Tusnady GE, Welker E
  TITLE     A method for characterizing Cas9 variants via a one-million target 
            sequence library of self-targeting sgRNAs.
  JOURNAL   Nucleic Acids Res. 2021 Jan 15. pii: 6101605. doi: 
            10.1093/nar/gkaa1220.
  PUBMED    33450024
REFERENCE   2  (bases 1 to 3060)
  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: "Nucleic 
            Acids Res. 2021 Jan 15. pii: 6101605. doi: 10.1093/nar/gkaa1220."
COMMENT     Sequence Label: pAT-9208
FEATURES             Location/Qualifiers
     source          1..3060
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    12..33
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(48..53,54..71,72..78)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    86..102
                     /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)."
     primer_bind     91..113
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     110..126
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     110..126
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_feature    182..200
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     promoter        204..238
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     terminator      256..283
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     misc_feature    488..506
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     primer_bind     complement(570..587)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(570..586)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(579..601)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(795..814)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     914..936
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(974..992)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1060..1164
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1165..1233,1234..2025)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1383..1402)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2196..2784
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2685..2704
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2938..2955
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
       61 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
      121 tatgaccatg attacgccaa gcttgcatgc aggcctctgc agtcgactcg tcggcagcgt
      181 cagatgtgta taagagacag atcttgacag ctagctcagt cctaggtata atgctagccg
      241 agacgcggcc gctgcagaag gccatcctga cggatggcct tttgagctct ggcaaacagc
      301 tattatgggt attatgggta ccggtgaatt cgatacaaat ctccgttgta ctttgtttcg
      361 cgcttggtat aatcgctggg gcaactttaa aaacgtctcg gggttagagc tagaaatagc
      421 aagttaacct aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
      481 ttgcatgctg tctcttatac acatctccga gcccacgaga ccatggatcc gatatctaga
      541 tgcattcgcg aggtaccgag ctcgaattca ctggccgtcg ttttacaacg tcgtgactgg
      601 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
      661 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
      721 gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
      781 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
      841 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
      901 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
      961 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     1021 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     1081 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     1141 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     1201 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     1261 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     1321 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     1381 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     1441 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     1501 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     1561 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     1621 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     1681 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     1741 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     1801 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     1861 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     1921 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     1981 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     2041 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     2101 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     2161 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     2221 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     2281 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     2341 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     2401 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     2461 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     2521 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     2581 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     2641 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     2701 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     2761 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     2821 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     2881 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     2941 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     3001 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
//
