LOCUS       sequence-478359-        8022 bp    DNA     circular SYN 14-JUL-2025
DEFINITION  Expression of Flag- and His-tagged LARP1delta327-496.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8022)
  AUTHORS   Shichino Y, Yamaguchi T, Kashiwagi K, Mito M, Takahashi M, Ito T, 
            Ingolia NT, Kuba K, Iwasaki S
  TITLE     eIF4A1 enhances LARP1-mediated translational repression during 
            mTORC1 inhibition.
  JOURNAL   Nat Struct Mol Biol. 2024 Oct;31(10):1557-1566. doi: 
            10.1038/s41594-024-01321-7. Epub 2024 May 21.
  PUBMED    38773334
REFERENCE   2  (bases 1 to 8022)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41594-024-01321-7"; journalName: "Nat Struct Mol Biol"; 
            date: "2024-10"; volume: "31"; issue: "10"; pages: "1557-1566"
COMMENT     Sequence Label: pcDNA5/FRT/TO-3xFLAG-His6-LARP1-d327-496
FEATURES             Location/Qualifiers
     source          1..8022
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      979..988
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             988..1053
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1054..1071
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1072..1083
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3974..3991)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3980..4204
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4337..4356)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4488..4535
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             4543..5565
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4543..5565
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    5695..5816
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5732..5751)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5786..5805
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5865..5881)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5865..5881)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5878..5900)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5889..5905
                     /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(5913..5919,5920..5937,5938..5943))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5958..5979
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6096..6113)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6267..6855)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6347..6366)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7026..7817,7818..7886))
                     /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     7649..7668
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7887..7991)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttagc caccatggac tacaaagacc atgacggtga ttataaagat
     1021 catgacatcg attacaagga tgacgatgac aagcatcatc atcatcatca tatcgaaggt
     1081 aggcatatgg ccactcaggt ggagccgctg ctgcctgggg gcgccacgct cttgcaggcc
     1141 gaagagcacg gggggctggt gaggaagaag ccgccgccgg cgcccgaggg caagggcgag
     1201 cccgggccaa acgacgtccg cgggggggag ccggacggca gcgctcggag accccggccg
     1261 ccctgcgcca agccgcacaa ggagggcacc gggcagcagg agcgcgagag cccgcggccg
     1321 ctgcagctgc ccggcgccga aggcccggcc atcagcgacg gggaggaggg cggcggcgag
     1381 ccaggcgctg gcggaggagc tgccggagcc gcgggcgcgg ggcgccggga cttcgtggaa
     1441 gcccccccgc ccaaggtgaa cccgtggact aagaacgcat tgccgccggt cctgaccacc
     1501 gtgaacggac agtccccccc agaacactct gctccagcca aggtggtgag ggcagctgtt
     1561 cctaaacagc gcaaaggcag caaggttggt gactttggag atgcaatcaa ttggcccaca
     1621 cctggagaga tagcccacaa gagtgttcag ccacagtccc acaagcctca gcctacccgt
     1681 aaactgccac ccaagaagga catgaaggaa caggagaaag gagaagggag tgatagtaag
     1741 gagagtccaa aaaccaaatc agatgaatca ggggaggaaa agaatggaga tgaggattgc
     1801 cagcgaggcg ggcagaagaa gaaaggaaac aaacacaagt gggttccatt acaaatagac
     1861 atgaagcctg aagtgcccag agagaaactg gcttcacgcc ccactcgccc accggagcct
     1921 agacacatac ctgccaatcg cggagagatc aaagggtctg agtctgccac ctacgtgccc
     1981 gtggcccccc ccaccccagc ctggcaacca gagatcaaac cggagcctgc ctggcacgac
     2041 caggatgaga catcgagtgt gaagtcccag cttctcaact gccctgaatt tgttccccgt
     2101 cagcactacc aaaaggagac agagtcggca cctggctctc ctcgtgcagt caccccagtg
     2161 ccaaccaaaa cagaggaggt cagcaaccta aagacactac ccaagggcct gtctgccagc
     2221 ctgcctgacc tggattctga gaactggatt gaagtgaaga agaggcctcg gccatcccca
     2281 gcacggccca agaagtcaga ggagtccaga ttttcccacc tgacctctct gcctcagcag
     2341 ctgccttccc agcagctgat gtccaaggat caggatgagc aagaggaact ggattttctg
     2401 tttgacgagg agatggagca gatggatggg cggaagaaca ccttcactgc ctggtctgat
     2461 gaggaatctg actatgagat tgatgacagg gatgtcaaca agatcctcat tgtcacccag
     2521 acaccacatt acatgcgccg gcacccaggg ggggaccgca caggcaacca cacctcgcgt
     2581 gccaagatga gcgccgaact ggccaaggtc attaatgatg gcctcttcta ctatgagcag
     2641 gacctgtggg ctgaaaagtt tgaacctgag tattcccaga tcaagcaaga agtcgagaac
     2701 ttcaaaaagg tcaatatgat cagccgggag cagtttgaca cactgacccc tgagccccct
     2761 gtggatccca accaggaagt tcctcctggg ccacctcggt tccagcaagt tcctacggat
     2821 gccctggcca acaagttgtt tggtgctcct gagccctcca ccatcgcccg ctctctacca
     2881 accactgtcc cagagtcacc aaactaccgc aacaccagga cccctcgcac tccccggaca
     2941 ccacagctca aagactcaag ccagacatca cggttttacc cagtggtgaa agaaggacgg
     3001 acactggatg ccaagatgcc tcgaaaaaga aagacaagac acagttcaaa cccacccttg
     3061 gagagccatg tgggctgggt gatggattcc cgtgagcaca ggccccgtac tgcttccatc
     3121 agctccagcc cctcagaagg gacgcctaca gttggcagct atggctgtac ccctcagtca
     3181 ttgcccaagt tccagcatcc ttcccatgaa ctgctcaagg aaaatggctt cacacaacac
     3241 gtctaccata agtatcgtag gcgctgcctt aatgagcgga aacgcttggg cattggccag
     3301 tctcaggaga tgaacacact cttccgcttc tggtccttct tcctccgaga tcacttcaac
     3361 aaaaagatgt atgaggagtt caagcagctg gctctggagg acgccaaaga aggctacaga
     3421 tatggtttgg agtgcctttt tcgatactac agttatggcc tggaaaagaa gttccggctg
     3481 gacatattca aggattttca ggaggaaacg gtgaaggact atgaagctgg ccaactgtat
     3541 gggctggaga agttctgggc cttcttgaaa tattccaaag ccaaaaattt ggacattgac
     3601 cccaaactgc aagaatacct cggcaaattc cgacgtcttg aagacttccg agtagatccc
     3661 cccatgggtg aggagggcaa ccacaagcga cactcagtgg tagcaggagg tggcggcggt
     3721 gagggcagga agcggtgccc ctcccagtct tccagcaggc ctgctgccat gatcagccaa
     3781 ccccctacac cacccaccgg ccagcctgtc cgggaagatg ccaaatggac aagccagcac
     3841 tcgaacacac agactttggg aaagtgatgg taccgagctc ggatccacta gtccagtgtg
     3901 gtggaattct gcagatatcc agcacagtgg cggccgctcg agtctagagg gcccgtttaa
     3961 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     4021 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     4081 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     4141 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     4201 atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt
     4261 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     4321 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     4381 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     4441 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtacctagaa gttcctattc
     4501 cgaagttcct attctctaga aagtatagga acttccttgg ccaaaaagcc tgaactcacc
     4561 gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag
     4621 ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc
     4681 ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt
     4741 gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg
     4801 acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa
     4861 ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt
     4921 agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg
     4981 cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac
     5041 gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac
     5101 tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac
     5161 aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac
     5221 gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc
     5281 tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc
     5341 cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct
     5401 tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca
     5461 caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat
     5521 agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagcacgt actacgagat
     5581 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     5641 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     5701 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     5761 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     5821 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     5881 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     5941 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     6001 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     6061 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     6121 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     6181 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     6241 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     6301 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     6361 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     6421 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     6481 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     6541 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     6601 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     6661 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     6721 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     6781 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     6841 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     6901 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     6961 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     7021 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     7081 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     7141 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     7201 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     7261 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     7321 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     7381 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     7441 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     7501 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     7561 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     7621 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     7681 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     7741 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     7801 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     7861 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     7921 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     7981 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
