LOCUS       sequence-136725-        4486 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pBT264-[miR-489].
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4486)
  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 4486)
  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-35
FEATURES             Location/Qualifiers
     source          1..4486
                     /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(2301..2319)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2340..2356)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2340..2356)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2353..2375)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2364..2380
                     /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(2388..2394,2395..2412,2413..2418))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2433..2454
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2571..2588)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2742..3330)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2822..2841)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3501..4292,4293..4361))
                     /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     4124..4143
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4362..4466)
                     /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 atgactcaga attccgttgc ggatccaagt gttatgtcta tactacttat
     1321 ttcagggggt taaataaaaa tatgattata tgttcacatt cagatagaaa tgagatatta
     1381 agggcatatt gtttttctga ataaacatca gttttcaaag ctttgcatat aaaccatctt
     1441 acatgatttt tcaacataca aacaaacaaa actcttaatt gttgtcccat gtagcagttt
     1501 agctgccgta tatgtgatgt cactcctaaa ggttcaagta aatggcgtca cacatacgac
     1561 caccaagctg ccaccaacag cagtagtcag cagcccaaac atcatcagtt tttttcagtc
     1621 gacaaatact ttgagtgcct ctcacatgtc aggcactctt ctaggttctg gaaattcagc
     1681 agtgaaccaa actggcaagc cacctgctct catagagctt acattcttga ttttatccac
     1741 actaatcatg gaatccacag gaaaggaatc tgatagatct tcagcctaaa gctttttccc
     1801 cgtatccccc caggtgtctg caggctcaaa gagcagcgag aagcgttcag aggaaagcga
     1861 tcccgtgcca ccttccccgt gcccgggctg tccccgcacg ctgccggctc ggggatgcgg
     1921 ggggagcgcc ggaccggagc ggagccccgg gcggctcgct gctgccccct agcgggggag
     1981 ggacgtaatt acatccctgg gggctttggg ggggggctgt ccccgtgcac tcagcctaaa
     2041 gctttttccc cgtatccccc caggtgtctg caggctcaaa gagcagcgag aagcgttcag
     2101 aggaaagcga tcccgtgcca ccttccccgt gcccgggctg tccccgcacg ctgccggctc
     2161 ggggatgcgg ggggagcgcc ggaccggagc ggagccccgg gcggctcgct gctgccccct
     2221 agcgggggag ggacgtaatt acatccctgg gggctttggg ggggggctgt ccccatttaa
     2281 atcagctcca gcttttgttc cctttagtga gggttaattt cgagcttggc gtaatcatgg
     2341 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     2401 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     2461 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     2521 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     2581 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     2641 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     2701 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     2761 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     2821 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     2881 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     2941 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     3001 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     3061 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     3121 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     3181 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     3241 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     3301 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     3361 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     3421 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     3481 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     3541 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     3601 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     3661 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     3721 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     3781 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     3841 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     3901 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     3961 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     4021 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     4081 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     4141 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     4201 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     4261 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     4321 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     4381 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     4441 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgc
//
