LOCUS       sequence-437113-        3080 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The dual-scaffold for making dual-CRISPR screening libraries or 
            dual-CRISPR plasmids..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3080)
  AUTHORS   Li Y, Tan M, Akkari-Henic A, Zhang L, Kip M, Sun S, Sepers JJ, Xu N,
            Ariyurek Y, Kloet SL, Davis RP, Mikkers H, Gruber JJ, Snyder MP, Li 
            X, Pang B
  TITLE     Genome-wide Cas9-mediated screening of essential non-coding 
            regulatory elements via libraries of paired single-guide RNAs.
  JOURNAL   Nat Biomed Eng. 2024 May 22. doi: 10.1038/s41551-024-01204-8.
  PUBMED    38778183
REFERENCE   2  (bases 1 to 3080)
  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 Biomed 
            Eng. 2024 May 22. doi: 10.1038/s41551-024-01204-8."
COMMENT     Sequence Label: pUC19-dual-scafffold-2.0 (pBP49)
FEATURES             Location/Qualifiers
     source          1..3080
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      533..1121
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1022..1041
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1275..1292
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1409..1430
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1445..1450,1451..1468,1469..1475)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1483..1499
                     /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     1488..1510
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1507..1523
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1507..1523
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_RNA        1575..1650
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_RNA        complement(1886..1961)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1987..2004)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1987..2003)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1996..2018)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2212..2231)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2331..2353
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2391..2409)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2477..2581
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2582..2650,2651..3080,1..362)
                     /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(2800..2819)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
       61 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
      121 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
      181 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
      241 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
      301 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
      361 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
      421 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
      481 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
      541 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
      601 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
      661 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
      721 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
      781 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
      841 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
      901 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
      961 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     1021 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     1081 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     1141 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     1201 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     1261 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     1321 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     1381 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
     1441 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
     1501 ttcacacagg aaacagctat gaccatgatt acgccaagct tgcatgcctg caggtcgact
     1561 ctagaggaag acgtgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat
     1621 caacttgaaa aagtggcacc gagtcggtgc ttttttcgtc tccacgtggt ggcgcccgga
     1681 tcccgactgc tccctgagtg tggctctttg ttctgtcaac gcccgacctt catcgcggcc
     1741 gattccttct gcggaccatg tcgtcctgat actttggcca tgtttccgtt gtaggagtga
     1801 agccacttgg ctttgcgccg tagttccaat gaaaaaccta tggactttgt ttaggatccg
     1861 gtagcatcag gacgagacga aaaaagcacc gactcggtgc cactttttca agttgataac
     1921 ggactagcct tattttaact tgctatttct agctctaaaa cacgtcttcg taccgagctc
     1981 gaattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     2041 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     2101 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt
     2161 tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg
     2221 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     2281 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     2341 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     2401 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     2461 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     2521 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     2581 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     2641 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     2701 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     2761 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     2821 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     2881 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     2941 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     3001 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     3061 tgatcgttgg gaaccggagc
//
