LOCUS       sequence-170533-        5536 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-terminal HA-tagged protein expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5536)
  AUTHORS   Yun YS, Kim KH, Tschida B, Sachs Z, Noble-Orcutt KE, Moriarity BS, 
            Ai T, Ding R, Williams J, Chen L, Largaespada D, Kim DH
  TITLE     mTORC1 Coordinates Protein Synthesis and Immunoproteasome Formation 
            via PRAS40 to Prevent Accumulation of Protein Stress.
  JOURNAL   Mol Cell. 2016 Feb 18;61(4):625-39. doi: 
            10.1016/j.molcel.2016.01.013. Epub 2016 Feb 11.
  PUBMED    26876939
REFERENCE   2  (bases 1 to 5536)
  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.2016.01.013"; journalName: "Mol Cell"; date: 
            "2016-02-18- 18"; volume: "61"; issue: "4"; pages: "625-39"
COMMENT     Sequence Label: HA-PRAS40 F129A
FEATURES             Location/Qualifiers
     source          1..5536
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        14..393
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        394..597
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     548..568
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     594..618
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        828..846
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     828..845
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     859..878
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      963..972
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             975..1001
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      1836..1845
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1849..1983
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1886..1905)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1940..1959
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2052..2409
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2052..2243
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2075..2095)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2260..2395
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2429..2446)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2429..2445)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2438..2460)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2658..3113
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2745..2764)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2955..2976
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3130..3149)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3249..3271
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3309..3327)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3395..3499
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3500..3568,3569..4360)
                     /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(3718..3737)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4531..5119
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5020..5039
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5273..5290
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5407..5428
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5443..5448,5449..5466,5467..5473)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5481..5497
                     /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)."
     primer_bind     5486..5508
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5505..5521
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5505..5521
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca
       61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
      601 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
      661 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt
      721 gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta
      781 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac
      841 tatagaataa catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc
      901 acctcggttc tatcgattga attcccgaac cgacagtcgg tctcttcacc aaggccattc
      961 gcgccaccat gggctacccc tatgatgtgc ctgactacgc agatctcaat ggtggtggtg
     1021 gtgggtcgac gatggcgtcg ggtcgccctg aggagctgtg ggaggccgtg gtgggggccg
     1081 ctgagcgctt ccgggcccgg actggcacgg agctggtgct gctgaccgcg gccccgccgc
     1141 caccaccccg cccgggcccc tgtgcctatc ctgcccatgg tcgaggagcc ctggcggagg
     1201 cagcgcgccg ttgcctccac gacatcgcac tggcccacag ggctgccact gctgctcggc
     1261 ctcctgcgcc cccaccagca ccacagccac ccagtcccac acccagccca ccccggccta
     1321 ccctggccag agaggacaac gaggaggacg aggatgagcc cacagagaca gagacctccg
     1381 gggagcagct gggcattagt gataatggag ggctcgctgt gatggatgag gacgccaccc
     1441 tccaggacct tccccccttc tgtgagtcag accccgagag tacagatgat ggcagcctga
     1501 gcgaggagac ccccgccggc ccccccacct gctcagtgcc cccagcctca gccctaccca
     1561 cacagcagta cgccaagtcc ctgcctgtgt ctgtgcccgt ctggggcttc aaggagaaga
     1621 ggacagaggc gcggtcatca gatgaggaga atgggccgcc ctcttcgccc gacctggacc
     1681 gcatcgcggc gagcatgcgc gcgctggtgc tgcgagaggc cgaggacacc caggtcttcg
     1741 gggacctgcc acggccgcgg cttaacacca gcgacttcca gaagctgaag cggaaatatt
     1801 gagcggccgc taagtaagta aggatcccca gcttggccgc catggcccaa cttgtttatt
     1861 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     1921 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
     1981 atcgggaatt aattcggcgc agcaccatgg cctgaaataa cctctgaaag aggaacttgg
     2041 ttaggtacct tctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga
     2101 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2161 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2221 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     2281 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     2341 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     2401 ttttgcaaaa agctgttaac agcttggcac tggccgtcgt tttacaacgt cgtgactggg
     2461 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     2521 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     2581 aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     2641 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2701 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     2761 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2821 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     2881 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     2941 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggg
     3001 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     3061 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
     3121 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     3181 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     3241 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     3301 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     3361 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     3421 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     3481 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     3541 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     3601 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     3661 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     3721 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     3781 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     3841 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     3901 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     3961 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     4021 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     4081 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     4141 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     4201 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     4261 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     4321 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     4381 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     4441 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     4501 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     4561 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     4621 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     4681 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     4741 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     4801 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     4861 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     4921 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4981 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     5041 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     5101 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     5161 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     5221 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     5281 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     5341 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     5401 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
     5461 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
     5521 catgattacg aattaa
//
