LOCUS       sequence-290332-        4576 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of shRNA2-HsNot9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4576)
  AUTHORS   Chen Y, Boland A, Kuzuoglu-Ozturk D, Bawankar P, Loh B, Chang CT, 
            Weichenrieder O, Izaurralde E
  TITLE     A DDX6-CNOT1 complex and W-binding pockets in CNOT9 reveal direct 
            links between miRNA target recognition and silencing.
  JOURNAL   Mol Cell. 2014 Jun 5;54(5):737-50. doi: 
            10.1016/j.molcel.2014.03.034. Epub 2014 Apr 24.
  PUBMED    24768540
REFERENCE   2  (bases 1 to 4576)
  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: 
            "10.1016/j.molcel.2014.03.034"; journalName: "Mol Cell"; date: 
            "2014-06-5- 5"; volume: "54"; issue: "5"; pages: "737-50"
COMMENT     Sequence Label: pSUPERpuro-BglII-HsNot9shRNA2_W
FEATURES             Location/Qualifiers
     source          1..4576
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /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"
     promoter        712..926
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     847..866
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     primer_bind     complement(1000..1016)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1001..1017)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        1018..1347
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1198..1333
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             1444..2043
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1444..1463)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1940..1960
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    2273..2354
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2310..2329)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2388..2408)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2388..2406)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2427..2443)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2427..2443)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2440..2462)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2451..2467
                     /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(2475..2481,2482..2499,2500..2505))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2520..2541
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2658..2675)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2829..3417)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2909..2928)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3588..4379,4380..4448))
                     /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     4211..4230
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4449..4553)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctcca
      661 ccgcggtggc ggccgctcta gactagtgga tcccccgggc tgcaggaatt cgaacgctga
      721 cgtcatcaac ccgctccaag gaatcgcggg cccagtgtca ctaggcggga acacccagcg
      781 cgcgtgcgcc ctggcaggaa gatggctgtg agggacaggg gagtggcgcc ctgcaatatt
      841 tgcatgtcgc tatgtgttct gggaaatcac cataaacgtg aaatgtcttt ggatttggga
      901 atcttataag ttctgtatga gaccacagat ccccgatcta tcagtggatc aatttcaaga
      961 gaattgatcc actgatagat cttttttgga aaagcttatc gataccgtcg acctcgagtg
     1021 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     1081 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     1141 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     1201 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     1261 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     1321 ttttttggag gcctaggctt ttgcaaaaag ctagcttgca tgcctgcagg tcggccgcca
     1381 cgaccggtgc cgccaccatc ccctgaccca cgcccctgac ccctcacaag gagacgacct
     1441 tccatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt cccccgggcc
     1501 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgacccg
     1561 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     1621 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     1681 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     1741 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag
     1801 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg
     1861 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc
     1921 ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     1981 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     2041 tgacgcccgc cccacgaccc gcagcgcccg accgaaagga gcgcacgacc ccatggctcc
     2101 gaccgaagcc acccggggcg gccccgccga ccccgcaccc gcccccgagg cccaccgact
     2161 ctagaggatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct
     2221 cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt
     2281 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     2341 atttttttca ctgcctcgag ggggggcccg gtacccagct tttgttccct ttagtgaggg
     2401 ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     2461 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     2521 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     2581 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     2641 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     2701 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     2761 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     2821 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     2881 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     2941 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     3001 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     3061 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     3121 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     3181 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     3241 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     3301 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     3361 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     3421 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     3481 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     3541 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     3601 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     3661 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     3721 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     3781 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     3841 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     3901 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     3961 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     4021 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     4081 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     4141 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     4201 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     4261 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     4321 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     4381 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     4441 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     4501 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     4561 ccccgaaaag tgccac
//
