LOCUS       sequence-291277-        7654 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of HsNot9-V181E-shRNA2res.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7654)
  AUTHORS   Sgromo A, Raisch T, Backhaus C, Keskeny C, Alva V, Weichenrieder O, 
            Izaurralde E
  TITLE     Drosophila Bag-of-marbles directly interacts with the CAF40 subunit 
            of the CCR4-NOT complex to elicit repression of mRNA targets.
  JOURNAL   RNA. 2018 Mar;24(3):381-395. doi: 10.1261/rna.064584.117. Epub 2017 
            Dec 18.
  PUBMED    29255063
REFERENCE   2  (bases 1 to 7654)
  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: 
            "10.1261/rna.064584.117"; journalName: "RNA"; date: "2018-03"; 
            volume: "24"; issue: "3"; pages: "381-395"
COMMENT     Sequence Label: pCIneo-V5-SBP-MBP-HsNot9-V181E-shRNA2res_AG
FEATURES             Location/Qualifiers
     source          1..7654
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..729
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     680..700
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     726..750
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          890..1022
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1005..1024
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1067..1085
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1098..1107
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1107..1148
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1170..1283
                     /codon_start=1
                     /product="streptavidin-binding peptide"
                     /label=SBP
                     /note="selected from a peptide library; binds streptavidin 
                     with nanomolar affinity (Keefe et al., 2001)"
                     /translation="MDEKTTGWGGGHVVEGLAGELEQLRARLEHHPQGQREP"
     regulatory      2265..2274
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3358..3479
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3369..3388)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3423..3442
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3665..4120
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3752..3771)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3962..3983
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4237..4594
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4237..4428
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4260..4280)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4445..4580
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4645..5439
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4699..4718)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5309..5328
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5503..5551
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(5592..5611)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5711..5733
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5771..5789)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5857..5961
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5962..6030,6031..6822)
                     /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(6180..6199)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6993..7581
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7482..7501
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactg
      661 cgatcgcccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
      721 agcagagctc gtttagtgaa ccgtcagatc actagaagct ttattgcggt agtttatcac
      781 agttaaattg ctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcagt
      841 gactctctta aggtagcctt gcagaagttg gtcgtgaggc actgggcagg taagtatcaa
      901 ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac agagaagact
      961 cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct ttctctccac
     1021 aggtgtccac tcccagttca attacagctc ttaaggctag agtacttaat acgactcact
     1081 ataggctagc aattgcggcc accatgggta agcctatccc taaccctctc ctcggtctcg
     1141 attctacgcc cggaagcggt agcggtacca tggacgagaa gaccaccggc tgggggggcg
     1201 gccacgtggt ggagggcctg gccggcgagc tggagcagct gcgggccagg ctggagcacc
     1261 accctcaggg ccagcgggag ccctccggcg gctgcaagcc cctcgacatg aaaatcgaag
     1321 aaggtaaact ggtaatctgg attaacggcg ataaaggcta taacggtctc gctgaagtcg
     1381 gtaagaaatt cgagaaagat accggaatta aagtcaccgt tgagcatccg gataaactgg
     1441 aagagaaatt cccgcaggtt gcggcaactg gcgatggccc tgacattatc ttctgggcac
     1501 acgaccgctt tggtggctac gctcaatctg gcctgttggc tgaaatcacc ccggacaaag
     1561 cgttccagga caagctgtat ccgtttacct gggatgccgt acgttacaac ggcaagctga
     1621 ttgcttaccc gatcgctgtt gaagcgttat cgctgattta taacaaagat ctgctgccga
     1681 acccgccaaa aacctgggaa gagatcccgg cgctggataa agaactgaaa gcgaaaggta
     1741 agagcgcgct gatgttcaac ctgcaagaac cgtacttcac ctggccgctg attgctgctg
     1801 acgggggtta tgcgttcaag tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg
     1861 ataacgctgg cgcgaaagcg ggtctgacct tcctggttga cctgattaaa agcaaacaca
     1921 tgaatgcaga caccgattac tccatcgcag aagctgcctt taataaaggc gaaacagcga
     1981 tgaccatcaa cggcccgtgg gcatggtcca acatcgacac cagcaaagtg aattatggtg
     2041 taacggtact gccgaccttc aagggtcaac catccaaacc gttcgttggc gtgctgagcg
     2101 caggtattaa cgccgccagt ccgaacaaag agctggcgaa agagttcctc gaaaactatc
     2161 tgctgactga tgaaggtctg gaagcggtta ataaagacaa accgctgggt gccgtagcgc
     2221 tgaagtctta cgaggaagag ttggcgaaag atccacgtat tgccgccacc atggaaaacg
     2281 cccagaaagg tgaaatcatg ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc
     2341 gtactgcggt gatcaacgcc gccagcggtc gtcagactgt cgatgaagcc ctgaaagacc
     2401 ctccactcga gatgcacagc ctggcgacgg ctgcgcctgt gcctactaca ctggcacaag
     2461 tggatagaga aaaaatttac caatggatta acgagctgtc cagtcctgag actagggaaa
     2521 atgctttgct ggagctaagt aagaagcgag aatctgttcc tgaccttgca cccatgctgt
     2581 ggcattcatt tggtactatt gcagcacttt tacaggaaat tgtaaatatt tatccatcta
     2641 tcaacccacc caccttgaca gcacaccagt ctaacagagt ttgcaatgct ctggcattac
     2701 tgcaatgtgt agcatcacat ccagaaacca ggtcagcgtt tctcgcagca cacatcccac
     2761 tttttttgta cccctttttg cacactgtca gcaaaacacg tccctttgag tatctccggc
     2821 tcaccagcct tggagttatt ggggccctgg tgaaaacaga tgaacaagaa gtaatcaact
     2881 ttttattaac aacagaaatt atccctttat gtttgcgaat tatggaatct ggaagtgaac
     2941 tttctaaaac agaagccaca ttcatcctcc agaagatctt gttagatgac actggtttgg
     3001 cttatatatg tcagacgtat gagcgtttct cccatgttgc catgatcttg ggtaagatgg
     3061 tcctgcagct atccaaagag ccttctgccc gtctgctgaa gcatgtagtg agatgttacc
     3121 ttcgactttc agataacccc agggcacgtg aagcactcag acagtgcctc cctgaccagc
     3181 tgaaagacac aaccttcgcc caggtgctaa aagatgacac caccacgaaa cgctggcttg
     3241 cacaactggt gaagaacctg caagagggcc aggtcaccga tccccggggt atccccctgc
     3301 cccctcagtg agcggccgct tccctttagt gagggttaat gcttcgagca gacatgataa
     3361 gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt
     3421 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta
     3481 acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggagatgtgg gaggtttttt
     3541 aaagcaagta aaacctctac aaatgtggta aaatccgata aggatcgatc cgggctggcg
     3601 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     3661 atggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     3721 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     3781 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     3841 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     3901 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     3961 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     4021 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     4081 tttaacgcga attttaacaa aatattaacg cttacaattt cctgatgcgg tattttctcc
     4141 ttacgcatct gtgcggtatt tcacaccgca tacgcggatc tgcgcagcac catggcctga
     4201 aataacctct gaaagaggaa cttggttagg taccttctga ggcggaaaga accagctgtg
     4261 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     4321 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     4381 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     4441 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     4501 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     4561 aggaggcttt tttggaggcc taggcttttg caaaaagctt gattcttctg acacaacagt
     4621 ctcgaactta aggctagagc caccatgatt gaacaagatg gattgcacgc aggttctccg
     4681 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     4741 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     4801 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
     4861 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     4921 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     4981 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     5041 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     5101 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     5161 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     5221 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     5281 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     5341 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     5401 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     5461 tgaccgacca agcgacgccc aacctgccat cacgatggcc gcaataaaat atctttattt
     5521 tcattacatc tgtgtgttgg ttttttgtgt gaatcgatag cgataaggat ccgcgtatgg
     5581 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     5641 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     5701 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     5761 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     5821 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     5881 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     5941 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     6001 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     6061 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     6121 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     6181 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     6241 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     6301 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     6361 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     6421 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     6481 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     6541 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     6601 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     6661 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     6721 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     6781 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     6841 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     6901 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     6961 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     7021 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     7081 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     7141 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     7201 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     7261 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     7321 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     7381 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     7441 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     7501 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     7561 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     7621 tgctggcctt ttgctcacat ggctcgacag atct
//
