LOCUS       sequence-356642-        6402 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of HsRck_296-472-10xmut.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6402)
  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 6402)
  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: pcDNA3.1-MS2-HA-HsRck_296-472-10xmut_AC
FEATURES             Location/Qualifiers
     source          1..6402
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      661..670
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1099..1125
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(1721..1739)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1722..1739)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1759..1776)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1765..1989
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2035..2463
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2122..2141)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2332..2353
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2472..2492)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2477..2806
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2657..2792
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2719..2738
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2873..3667
                     /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(2927..2946)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3537..3556
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3841..3962
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3878..3897)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3932..3951
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4011..4027)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4011..4027)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4024..4046)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4035..4051
                     /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(4059..4065,4066..4083,4084..4089))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4104..4125
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4242..4259)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4413..5001)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4493..4512)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5172..5963,5964..6032))
                     /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     5795..5814
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6033..6137)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6212..6231)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 gccaccatgg cttctaactt tactcagttc gttctcgtcg acaatggcgg aactggcgac
      721 gtgactgtcg ccccaagcaa cttcgctaac ggggtcgctg aatggatcag ctctaactcg
      781 cgatcacagg cttacaaagt aacctgtagc gttcgtcaga gctctgcgca gaatcgcaaa
      841 tacaccatca aagtcgaggt gcctaaagtg gcaacccaga ctgttggtgg tgaagagctt
      901 cctgtagccg gatggagatc ttacttaaat atggaactaa ccattccaat tttcgccacg
      961 aattccgact gcgagcttat tgttaaggca atgcaaggtc tcctaaaaga tggaaacccg
     1021 attccctcgg ccatcgcggc caactccggc atctacggag gtggaggtgg atctggtgga
     1081 tctcctccac atatggctta cccatacgat gttccagatt acgctcctcc aaagcttgga
     1141 tccgaattcc cgggtcgact cgagaaggga gtaacccagt actacgcata tgtaactgag
     1201 cgcgcaaaag tagcgtgcct caacgcactt ttctccgcac ttgcaatagc ccagtcgatc
     1261 attttctgta actcctctca ggcagttgaa ttgctagccg cggcgatttc tcaactgggt
     1321 tattcttgct tctatattca tgctaaaatg aggcaggaac atcgaaatcg tgtatttcat
     1381 gatttccgaa atggcttatg cgccaatctt gtttgcactg atctgtttac ccgaggtatt
     1441 gatatacaag ctgtgaatgt ggtaataaac tttgatttcc caaagctggc agagacctat
     1501 ctccatcgta ttggaagatc aggtcgcttt ggtcatcttg gcttagccat caacttgatc
     1561 acatatgatg atcgcttcaa cctgaaaagt attgaggagc agctgggaac agaaattaaa
     1621 cctattccga gcaacattga taagagcctg tatgtggcag aataccacag cgagcctgta
     1681 gaagatgaga aaccttaagc ggccgcctag agggccctat tctatagtgt cacctaaatg
     1741 ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     1801 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     1861 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     1921 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     1981 gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc
     2041 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     2101 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     2161 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     2221 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     2281 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     2341 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     2401 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     2461 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     2521 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     2581 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     2641 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     2701 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt
     2761 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc
     2821 ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga
     2881 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     2941 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     3001 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga
     3061 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     3121 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     3181 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     3241 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     3301 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     3361 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga
     3421 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     3481 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     3541 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     3601 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     3661 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca
     3721 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     3781 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     3841 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     3901 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     3961 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     4021 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     4081 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     4141 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     4201 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     4261 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     4321 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     4381 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     4441 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     4501 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     4561 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     4621 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     4681 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     4741 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     4801 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     4861 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     4921 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     4981 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     5041 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     5101 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     5161 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     5221 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     5281 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     5341 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     5401 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     5461 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     5521 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     5581 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     5641 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     5701 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     5761 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     5821 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     5881 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     5941 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     6001 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     6061 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     6121 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga
     6181 gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     6241 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
     6301 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
     6361 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg tt
//
