LOCUS       sequence-339302-        4569 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of Hs4E-T-shRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4569)
  AUTHORS   Kuzuoglu-Ozturk D, Bhandari D, Huntzinger E, Fauser M, Helms S, 
            Izaurralde E
  TITLE     miRISC and the CCR4-NOT complex silence mRNA targets independently 
            of 43S ribosomal scanning.
  JOURNAL   EMBO J. 2016 Jun 1;35(11):1186-203. doi: 10.15252/embj.201592901. 
            Epub 2016 Mar 23.
  PUBMED    27009120
REFERENCE   2  (bases 1 to 4569)
  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: 
            "10.15252/embj.201592901"; journalName: "EMBO J"; date: "2016-06-1- 
            1"; volume: "35"; issue: "11"; pages: "1186-203"
COMMENT     Sequence Label: pSUPERpuro-BglII-Hs4E-T_AC
FEATURES             Location/Qualifiers
     source          1..4569
                     /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"
     primer_bind     complement(285..301)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(286..302)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        303..632
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      483..618
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             729..1328
                     /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(729..748)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1225..1245
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1558..1639
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1595..1614)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(1672..1690)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1711..1727)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1711..1727)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1724..1746)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1735..1751
                     /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(1759..1765,1766..1783,1784..1789))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1804..1825
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1942..1959)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2113..2701)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2193..2212)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2872..3663,3664..3732))
                     /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     3495..3514
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3733..3837)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3863..4318)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4000..4021)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4212..4231
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4445..4467
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4459..4476
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4460..4476
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4483..4502
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4483..4501
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     4534..4550
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4534..4550
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
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 accacagatc ccccgaacaa gattatcgac
      241 ctattcaaga gataggtcga taatcttgtt ctttttaagc ttatcgatac cgtcgacctc
      301 gagtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
      361 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
      421 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
      481 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
      541 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
      601 gaggcttttt tggaggccta ggcttttgca aaaagctagc ttgcatgcct gcaggtcggc
      661 cgccacgacc ggtgccgcca ccatcccctg acccacgccc ctgacccctc acaaggagac
      721 gaccttccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtccccc
      781 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg
      841 acccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg
      901 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca
      961 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt
     1021 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc
     1081 ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg
     1141 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg
     1201 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg
     1261 tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg
     1321 gtgcctgacg cccgccccac gacccgcagc gcccgaccga aaggagcgca cgaccccatg
     1381 gctccgaccg aagccacccg gggcggcccc gccgaccccg cacccgcccc cgaggcccac
     1441 cgactctaga ggatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
     1501 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac
     1561 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     1621 aaagcatttt tttcactgcc tcgagggggg gcccggtacc agcttttgtt ccctttagtg
     1681 agggttaatt tcgagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     1741 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     1801 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     1861 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     1921 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     1981 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     2041 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     2101 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     2161 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     2221 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     2281 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     2341 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     2401 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     2461 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     2521 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     2581 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     2641 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     2701 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     2761 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     2821 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     2881 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     2941 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     3001 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     3061 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     3121 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     3181 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     3241 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     3301 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     3361 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     3421 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     3481 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     3541 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     3601 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     3661 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     3721 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     3781 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     3841 atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta
     3901 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     3961 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     4021 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     4081 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     4141 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     4201 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     4261 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc
     4321 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     4381 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     4441 ttttcccagt cacgacgttg taaaacgacg gccagtgaat tgtaatacga ctcactatag
     4501 ggcgaattgg agctccaccg cggtggcggc cgctctagaa ctagtggatc ccccgggctg
     4561 caggaattc
//
