LOCUS       sequence-462122-        3476 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A dual-targeting vector that contains PE3 system guide RNAs against 
            BFP (pegRNA(BG) and nsgRNA(BG)) with restriction enzyme sites for 
            insertion of target site guide RNAS (pegRNAs(TS) and nsgRNA(TS))..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3476)
  AUTHORS   Frisch C, Kostes WW, Galyon B, Whitman B, Tekel SJ, Standage-Beier 
            K, Srinivasan G, Wang X, Brafman DA
  TITLE     PINE-TREE enables highly efficient genetic modification of human 
            cell lines.
  JOURNAL   Mol Ther Nucleic Acids. 2023 Jul 17;33:483-492. doi: 
            10.1016/j.omtn.2023.07.007. eCollection 2023 Sep 12.
  PUBMED    37588683
REFERENCE   2  (bases 1 to 3476)
  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: "Mol Ther 
            Nucleic Acids."; date: "2023-07-17"; volume: "33:483-492. doi"; 
            pages: " 10.1016/j.omtn.2023.07.007. eCollection 2023 Sep 12"
COMMENT     Sequence Label: PE-pegRNA(BG)-nsgRNA(BG)
FEATURES             Location/Qualifiers
     source          1..3476
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(247..1038,1039..1107))
                     /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     870..889
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1108..1212)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1280..1298
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1336..1358)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1458..1477
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1671..1693
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1685..1702
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1686..1702
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1709..1957
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1979..2054
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2117..2365
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        2387..2462
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2562..2578)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2562..2578)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2575..2597)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2586..2602
                     /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)."
     promoter        complement(join(2610..2616,2617..2634,2635..2640))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2655..2676
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2793..2810)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(2964..3476,1..76))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3044..3063)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
       61 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
      121 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
      181 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
      241 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
      301 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
      361 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
      421 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
      481 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
      541 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
      601 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
      661 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
      721 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
      781 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
      841 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
      901 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
      961 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     1021 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     1081 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     1141 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     1201 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat
     1261 catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg
     1321 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     1381 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     1441 gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg
     1501 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgccatt cgccattcag
     1561 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc
     1621 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg
     1681 acgttgtaaa acgacggcca gtgaattcga gggcctattt cccatgattc cttcatattt
     1741 gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa
     1801 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt
     1861 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt
     1921 cttggcttta tatatcttgt ggaaaggacg aaacaccgct cgtgaccacc ctgacccagt
     1981 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
     2041 caccgagtcg gtgccacgcc gtaggtcagg gtggtcattt tttgttttct gcaatccggc
     2101 aaaaaagggc tctagagagg gcctatttcc catgattcct tcatatttgc atatacgata
     2161 caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca
     2221 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
     2281 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
     2341 tatcttgtgg aaaggacgaa acaccggacg tagccttcgg gcatgggttt tagagctaga
     2401 aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
     2461 gcttttttgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg tttttagcgc
     2521 gtgcgccaat tctgcaggca tgcaagcttg gcgtaatcat ggtcatagct gtttcctgtg
     2581 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     2641 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     2701 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     2761 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     2821 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     2881 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     2941 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     3001 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     3061 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     3121 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     3181 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     3241 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     3301 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     3361 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     3421 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccgg
//
