LOCUS       sequence-314905-        4423 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pBT264-mutmU6p-{2xBbsI}-sgRNA scaffold-{MfeI}.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4423)
  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 4423)
  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: pAWp100
FEATURES             Location/Qualifiers
     source          1..4423
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..24,25..31)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    39..55
                     /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     44..66
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     63..79
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     63..79
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        100..118
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     877..897
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     misc_RNA        978..1053
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     polyA_signal    1100..1181
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1137..1156)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1711..1730)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1712..1730)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1737..1754)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1737..1753)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1746..1768)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1895..2350
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1982..2001)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2192..2213
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2376..2480
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2481..2549,2550..3341)
                     /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(2699..2718)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3512..4100
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4001..4020
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4254..4271
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4388..4409
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
       61 cacaggaaac agctatgacc atgattacgc caagctcgaa attaaccctc actaaaggga
      121 acaaaagctg gagctgattt aaatggggac agcccccccc caaagccccc agggatgtaa
      181 ttacgtccct cccccgctag ggggcagcag cgagccgccc ggggctccgc tccggtccgg
      241 cgctcccccc gcatccccga gccggcagcg tgcggggaca gcccgggcac ggggaaggtg
      301 gcacgggatc gctttcctct gaacgcttct cgctgctctt tgagcctgca gacacctggg
      361 gggatacggg gaaaaagctt taggctgagt gcacggggac agcccccccc caaagccccc
      421 agggatgtaa ttacgtccct cccccgctag ggggcagcag cgagccgccc ggggctccgc
      481 tccggtccgg cgctcccccc gcatccccga gccggcagcg tgcggggaca gcccgggcac
      541 ggggaaggtg gcacgggatc gctttcctct gaacgcttct cgctgctctt tgagcctgca
      601 gacacctggg gggatacggg gaaaaagctt taggctgaag atctgatccg acgccgccat
      661 ctctaggccc gcgccggccc cctcgcacag acttgtggga gaagctcggc tactcccctg
      721 ccccggttaa tttgcatata atatttccta gtaactatag aggcttaatg tgcgataaaa
      781 gacagataat ctgttctttt taatactagc tacattttac atgataggct tggatttcta
      841 taagagatac aaatactaaa ttattatttt aaaaaacagc acaaaaggaa actcacccta
      901 actgtaaagt aattgtgtgt tttgagacta taaatatccc ttggagaaaa gccttcaccg
      961 ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt
     1021 tatcaacttg aaaaagtggc accgagtcgg tgcttttttg gatccgcaac ggaattcctg
     1081 acgcacaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa
     1141 tagcatcaca aatttcacaa ataaagcatt tttttcactg cctcgaatca ctagtgggga
     1201 cagccccccc ccaaagcccc cagggatgta attacgtccc tcccccgcta gggggcagca
     1261 gcgagccgcc cggggctccg ctccggtccg gcgctccccc cgcatccccg agccggcagc
     1321 gtgcggggac agcccgggca cggggaaggt ggcacgggat cgctttcctc tgaacgcttc
     1381 tcgctgctct ttgagcctgc agacacctgg ggggatacgg ggaaaaagct ttaggctgag
     1441 tgcacgggga cagccccccc ccaaagcccc cagggatgta attacgtccc tcccccgcta
     1501 gggggcagca gcgagccgcc cggggctccg ctccggtccg gcgctccccc cgcatccccg
     1561 agccggcagc gtgcggggac agcccgggca cggggaaggt ggcacgggat cgctttcctc
     1621 tgaacgcttc tcgctgctct ttgagcctgc agacacctgg ggggatacgg ggaaaaagct
     1681 ttaggctgag gcgcgcctgt acccaattcg ccctatagtg agtcgtatta caattcactg
     1741 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     1801 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     1861 tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     1921 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     1981 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     2041 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     2101 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     2161 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     2221 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     2281 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     2341 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     2401 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     2461 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     2521 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     2581 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     2641 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     2701 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     2761 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     2821 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     2881 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     2941 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     3001 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     3061 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     3121 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     3181 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     3241 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     3301 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     3361 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     3421 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     3481 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     3541 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     3601 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     3661 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     3721 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     3781 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3841 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     3901 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     3961 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     4021 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     4081 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     4141 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     4201 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     4261 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     4321 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     4381 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggc
//
