LOCUS       sequence-314899-        4344 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pBT264-mutH1p-{2xBbsI}-sgRNA scaffold-{MfeI}.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4344)
  AUTHORS   Zhou P, Chan BKC, Wan YK, Yuen CTL, Choi GCG, Li X, Tong CSW, Zhong 
            SSW, Sun J, Bao Y, Mak SYL, Chow MZY, Khaw JV, Leung SY, Zheng Z, 
            Cheung LWT, Tan K, Wong KH, Chan HYE, Wong ASL
  TITLE     A Three-Way Combinatorial CRISPR Screen for Analyzing Interactions 
            among Druggable Targets.
  JOURNAL   Cell Rep. 2020 Aug 11;32(6):108020. doi: 
            10.1016/j.celrep.2020.108020.
  PUBMED    32783942
REFERENCE   2  (bases 1 to 4344)
  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: "Cell Rep.";
            date: "2020-08-11"; volume: "32(6):108020. doi"; pages: " 
            10.1016/j.celrep.2020.108020"
COMMENT     Sequence Label: pAWp92
FEATURES             Location/Qualifiers
     source          1..4344
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..215
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     136..155
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     misc_RNA        252..327
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     polyA_signal    374..455
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(411..430)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(985..1004)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(986..1004)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1011..1028)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1011..1027)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1020..1042)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1169..1624
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1256..1275)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1466..1487
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1650..1754
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1755..1823,1824..2615)
                     /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(1973..1992)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2786..3374
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3275..3294
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3528..3545
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3662..3683
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3698..3703,3704..3721,3722..3728)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3736..3752
                     /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     3741..3763
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3760..3776
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3760..3776
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3797..3815
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 gaacgctgac gtcatcaacc cgctccaagg aatcgcgggc ccagtgtcac taggcgggaa
       61 cacccagcgc gcgtgcgccc tggcaggaag atggctgtga gggacagggg agtggcgccc
      121 tgcaatattt gcatgtcgct atgtgttctg ggaaatcacc ataaacgtga aatgtctttg
      181 gatttgggaa tcttataagt tctgtatgag accactcttc accgggtctt cctcgagtgc
      241 agcgaagacc tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
      301 cttgaaaaag tggcaccgag tcggtgcttt tttggatccg caacggaatt cgaacttgac
      361 aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
      421 cacaaatttc acaaataaag catttttttc actgcctcga atcactagtg gggacagccc
      481 ccccccaaag cccccaggga tgtaattacg tccctccccc gctagggggc agcagcgagc
      541 cgcccggggc tccgctccgg tccggcgctc cccccgcatc cccgagccgg cagcgtgcgg
      601 ggacagcccg ggcacgggga aggtggcacg ggatcgcttt cctctgaacg cttctcgctg
      661 ctctttgagc ctgcagacac ctggggggat acggggaaaa agctttaggc tgagtgcacg
      721 gggacagccc ccccccaaag cccccaggga tgtaattacg tccctccccc gctagggggc
      781 agcagcgagc cgcccggggc tccgctccgg tccggcgctc cccccgcatc cccgagccgg
      841 cagcgtgcgg ggacagcccg ggcacgggga aggtggcacg ggatcgcttt cctctgaacg
      901 cttctcgctg ctctttgagc ctgcagacac ctggggggat acggggaaaa agctttaggc
      961 tgaggcgcgc ctgtacccaa ttcgccctat agtgagtcgt attacaattc actggccgtc
     1021 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     1081 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     1141 cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     1201 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     1261 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     1321 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     1381 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     1441 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     1501 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     1561 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     1621 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     1681 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     1741 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     1801 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     1861 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     1921 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     1981 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     2041 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     2101 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     2161 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     2221 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     2281 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     2341 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     2401 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     2461 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     2521 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     2581 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     2641 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     2701 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     2761 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2821 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2881 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     2941 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     3001 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     3061 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     3121 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     3181 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     3241 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     3301 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3361 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3421 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     3481 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     3541 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     3601 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     3661 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     3721 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     3781 attacgccaa gctcgaaatt aaccctcact aaagggaaca aaagctggag ctgatttaaa
     3841 tggggacagc ccccccccaa agcccccagg gatgtaatta cgtccctccc ccgctagggg
     3901 gcagcagcga gccgcccggg gctccgctcc ggtccggcgc tccccccgca tccccgagcc
     3961 ggcagcgtgc ggggacagcc cgggcacggg gaaggtggca cgggatcgct ttcctctgaa
     4021 cgcttctcgc tgctctttga gcctgcagac acctgggggg atacggggaa aaagctttag
     4081 gctgagtgca cggggacagc ccccccccaa agcccccagg gatgtaatta cgtccctccc
     4141 ccgctagggg gcagcagcga gccgcccggg gctccgctcc ggtccggcgc tccccccgca
     4201 tccccgagcc ggcagcgtgc ggggacagcc cgggcacggg gaaggtggca cgggatcgct
     4261 ttcctctgaa cgcttctcgc tgctctttga gcctgcagac acctgggggg atacggggaa
     4321 aaagctttag gctgaagatc tttc
//
