LOCUS       sequence-292016-        3174 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA constant region and hU6 insert for programmed dual sgRNA 
            cloning. The sgRNA constant region contains a capture sequence (cs1)
            in the stem loop for direct capture Perturb-seq..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3174)
  AUTHORS   Replogle JM, Norman TM, Xu A, Hussmann JA, Chen J, Cogan JZ, Meer 
            EJ, Terry JM, Riordan DP, Srinivas N, Fiddes IT, Arthur JG, Alvarado
            LJ, Pfeiffer KA, Mikkelsen TS, Weissman JS, Adamson B
  TITLE     Combinatorial single-cell CRISPR screens by direct guide RNA capture
            and targeted sequencing.
  JOURNAL   Nat Biotechnol. 2020 Aug;38(8):954-961. doi: 
            10.1038/s41587-020-0470-y. Epub 2020 Mar 30.
  PUBMED    32231336
REFERENCE   2  (bases 1 to 3174)
  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: 
            "10.1038/s41587-020-0470-y"; journalName: "Nat Biotechnol"; date: 
            "2020-08"; volume: "38"; issue: "8"; pages: "954-961"
COMMENT     Sequence Label: pJR89
FEATURES             Location/Qualifiers
     source          1..3174
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     71..93
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     85..102
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     86..102
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     360..380
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     531..550
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(660..676)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(660..676)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(673..695)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    684..700
                     /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(708..714,715..732,733..738))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    753..774
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(891..908)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1062..1650)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1142..1161)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1821..2612,2613..2681))
                     /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     2444..2463
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2682..2786)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2854..2872
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2910..2932)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3032..3051
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg
       61 ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgcaacgcg atgacgatgg
      121 atagcgattc atcgatgagc tgacccgatc gccgccgccg gagggttgcg tttgagacgg
      181 gcgacagatc gtctcaagag ctaagcacaa gagtgcatag caagttgaaa taaggctagt
      241 ccgtttacaa cttggccgct ttaaggccgg tcctagcaag gccaagtggc acccgagtcg
      301 ggtgcttttt ttgctcgaat ctacactcag ctatggcgcg ccccaaggtc gggcaggaag
      361 agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga
      421 taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa
      481 agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata
      541 tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaagcca
      601 gaaaggagac gatcagttct ggaccagcga gctgtgctgc gactcgtggc gtaatcatgg
      661 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
      721 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
      781 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
      841 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
      901 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
      961 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     1021 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     1081 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     1141 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     1201 tcgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     1261 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     1321 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     1381 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     1441 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     1501 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     1561 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     1621 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     1681 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     1741 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     1801 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     1861 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     1921 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     1981 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     2041 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     2101 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     2161 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     2221 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     2281 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     2341 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     2401 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     2461 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     2521 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     2581 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     2641 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactctacct ttttcaatat
     2701 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     2761 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     2821 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     2881 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     2941 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     3001 gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg
     3061 caccaaatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc
     3121 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctca
//
