LOCUS       sequence-133542-        4458 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pBT264-[let-7e/miR-99b cluster].
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4458)
  AUTHORS   Wong AS, Choi GC, Cheng AA, Purcell O, Lu TK
  TITLE     Massively parallel high-order combinatorial genetics in human cells.
  JOURNAL   Nat Biotechnol. 2015 Sep;33(9):952-61. doi: 10.1038/nbt.3326. Epub 
            2015 Aug 17.
  PUBMED    26280411
REFERENCE   2  (bases 1 to 4458)
  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/nbt.3326"; 
            journalName: "Nat Biotechnol"; date: "2015-09"; volume: "33"; issue:
            "9"; pages: "952-61"
COMMENT     Sequence Label: pAWSV-5
FEATURES             Location/Qualifiers
     source          1..4458
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(33..461)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(143..164)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     355..374
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     588..610
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     602..619
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     603..619
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    1175..1256
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     1200..1219
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(2273..2291)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2312..2328)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2312..2328)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2325..2347)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2336..2352
                     /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(2360..2366,2367..2384,2385..2390))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2405..2426
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2543..2560)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2714..3302)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2794..2813)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3473..4264,4265..4333))
                     /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     4096..4115
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4334..4438)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
       61 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
      121 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
      181 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
      241 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
      301 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
      361 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
      421 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
      481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      541 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      601 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tgggtacagg
      661 cgcgcctcag cctaaagctt tttccccgta tccccccagg tgtctgcagg ctcaaagagc
      721 agcgagaagc gttcagagga aagcgatccc gtgccacctt ccccgtgccc gggctgtccc
      781 cgcacgctgc cggctcgggg atgcgggggg agcgccggac cggagcggag ccccgggcgg
      841 ctcgctgctg ccccctagcg ggggagggac gtaattacat ccctgggggc tttggggggg
      901 ggctgtcccc gtgcactcag cctaaagctt tttccccgta tccccccagg tgtctgcagg
      961 ctcaaagagc agcgagaagc gttcagagga aagcgatccc gtgccacctt ccccgtgccc
     1021 gggctgtccc cgcacgctgc cggctcgggg atgcgggggg agcgccggac cggagcggag
     1081 ccccgggcgg ctcgctgctg ccccctagcg ggggagggac gtaattacat ccctgggggc
     1141 tttggggggg ggctgtcccc actagtgatt cgaggcagtg aaaaaaatgc tttatttgtg
     1201 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     1261 acaacaattg tttggagtga attccgttgc ggatccgaca cagaagccac agagacacag
     1321 cagagatgga gacagacagg gaaggaaaac agatttcagg ggaaggaggg gatgcaggga
     1381 caaggacaga aaagagcccg gctcttcctc ccagggtccc tggggcagcc cagtggggct
     1441 aagggtccct gagtggggct gggggtcccc gccgggcccc gtcccgtgca gggcgcagcc
     1501 tggggaaagc taggaggccg tatagtgatc tccttgggtg tcctcctcaa ctatacaacc
     1561 tcctacctca gcccgggggg cgcggcaggt ggacagaccc gacagacaga cagacaggga
     1621 ccaggaggac cagggatgag ggggagggcc ggggaggccc cagccgcgat ggtgagcccc
     1681 cgacacggac ccacagacac gagcttgtgt gcggcgaagg ccccgcaagg tcggttctac
     1741 gggtggagat cttcagccta aagctttttc cccgtatccc cccaggtgtc tgcaggctca
     1801 aagagcagcg agaagcgttc agaggaaagc gatcccgtgc caccttcccc gtgcccgggc
     1861 tgtccccgca cgctgccggc tcggggatgc ggggggagcg ccggaccgga gcggagcccc
     1921 gggcggctcg ctgctgcccc ctagcggggg agggacgtaa ttacatccct gggggctttg
     1981 ggggggggct gtccccgtgc actcagccta aagctttttc cccgtatccc cccaggtgtc
     2041 tgcaggctca aagagcagcg agaagcgttc agaggaaagc gatcccgtgc caccttcccc
     2101 gtgcccgggc tgtccccgca cgctgccggc tcggggatgc ggggggagcg ccggaccgga
     2161 gcggagcccc gggcggctcg ctgctgcccc ctagcggggg agggacgtaa ttacatccct
     2221 gggggctttg ggggggggct gtccccattt aaatcagctc cagcttttgt tccctttagt
     2281 gagggttaat ttcgagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     2341 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     2401 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     2461 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     2521 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     2581 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     2641 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     2701 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     2761 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     2821 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     2881 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     2941 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     3001 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     3061 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     3121 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     3181 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     3241 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     3301 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     3361 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     3421 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     3481 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     3541 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     3601 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     3661 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     3721 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     3781 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     3841 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     3901 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     3961 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     4021 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     4081 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     4141 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     4201 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     4261 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     4321 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     4381 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     4441 catttccccg aaaagtgc
//
