LOCUS       sequence-314413-        7062 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-terminal HA-tagged Mios cDNA expression vector for transfections.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7062)
  AUTHORS   Orozco JM, Krawczyk PA, Scaria SM, Cangelosi AL, Chan SH, Kunchok T,
            Lewis CA, Sabatini DM
  TITLE     Dihydroxyacetone phosphate signals glucose availability to mTORC1.
  JOURNAL   Nat Metab. 2020 Sep;2(9):893-901. doi: 10.1038/s42255-020-0250-5. 
            Epub 2020 Jul 27.
  PUBMED    32719541
REFERENCE   2  (bases 1 to 7062)
  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: 
            "10.1038/s42255-020-0250-5"; journalName: "Nat Metab"; date: 
            "2020-09"; volume: "2"; issue: "9"; pages: "893-901"
COMMENT     Sequence Label: HA-mios in pRK5
FEATURES             Location/Qualifiers
     source          1..7062
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     201..225
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        435..453
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     435..452
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     466..485
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             complement(1294..1305)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             2922..2948
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      2969..2978
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2982..3116
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3019..3038)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3073..3092
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3185..3542
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3185..3376
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3208..3228)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3393..3528
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3562..3579)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3562..3578)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3571..3593)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3791..4246
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3878..3897)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4088..4109
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4263..4282)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4382..4404
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4442..4460)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4528..4632
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4633..4701,4702..5493)
                     /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(4851..4870)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5664..6252
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6153..6172
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6406..6423
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6540..6561
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6576..6581,6582..6599,6600..6606)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6614..6630
                     /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     6619..6641
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6638..6654
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6638..6654
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        6683..7062
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat
      241 ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccg cggccgggaa
      301 cggtgcattg gaacgcggat tccccgtgcc aagagtgacg taagtaccgc ctatagagtc
      361 tataggccca cccccttggc ttcgttagaa cgcggctaca attaatacat aaccttatgt
      421 atcatacaca tacgatttag gtgacactat agaataacat ccactttgcc tttctctcca
      481 caggtgtcca ctcccaggtc caactgcacc tcggttctat cgattgaatt cgagttgtac
      541 ttacaagcct tggtcaagat cataactcaa agttcaaaga tttgatagga aaagagtttg
      601 ttccaaaaca tgcacgacaa tgtaataccc ttgcctggaa tccactggat agtaactggc
      661 tagctgctgg tttagataag cacagagctg acttttcagt gctaatatgg gatatctgca
      721 gcaaatatac tcctgatata gttcccatgg aaaaagtgaa actttcagca ggtgaaactg
      781 aaacaacatt attagtaaca aaaccacttt atgagttagg acagaatgat gcttgtctgt
      841 ctctttgttg gcttccacga gaccagaaac ttctccttgc tggtatgcat cgtaacctag
      901 ctatatttga tcttcggaat acaagccaaa agatgttcgt aaatacaaaa gctgttcagg
      961 gtgtgacggt agacccatat ttccacgatc gtgttgcttc cttctatgaa ggtcaggttg
     1021 caatatggga tcttagaaaa tttgagaagc cagttttgac attgactgag caaccaaaac
     1081 ccttaacaaa agtagcatgg tgtcccacta ggactggtct acttgccact ttaacaaggg
     1141 atagtaatat tattagattg tatgatatgc agcatacacc cactcccatt ggggatgaaa
     1201 ctgaacccac aataattgaa agaagtgtgc aaccttgtga caattacatt gcttcctttg
     1261 cgtggcatcc aacaagtcaa aatcgaatga tagttgtaac tcccaaccga acaatgtcag
     1321 acttcactgt ttttgaaagg atatctcttg cctggagccc aattacatct ttaatgtggg
     1381 cttgtggtcg tcatttatat gaatgtacgg aagaagaaaa tgataattct ttagaaaaag
     1441 atatagcaac gaagatgcgt cttcgggctt tatcaaggta tggacttgat acagagcagg
     1501 tgtggaggaa ccacatttta gctggaaatg aagatccaca gctcaagtca ctctggtata
     1561 ctctgcactt tatgaagcaa tacacagaag atatggatca gaaatctcca ggcaacaaag
     1621 gatcattggt ttatgcagga attaaatcaa ttgtaaagtc atcgttggga atggtggaaa
     1681 gcagcagaca taattggagt gggttggata agcaaagtga tattcaaaat ttaaatgaag
     1741 agagaatctt agctttacag ctttgtgggt ggataaagaa aggaacggat gtagacgtgg
     1801 ggccattttt gaactccctt gtacaagaag gggaatggga aagagctgct gctgtggcat
     1861 tgttcaactt ggatattcgc cgagcaatcc aaatcctgaa tgaaggggca tcttctgaaa
     1921 aaggagatct gaatctcaat gtggtagcaa tggctttatc gggttatacg gatgagaaga
     1981 actccctttg gagagaaatg tgtagcacac tgcgattaca gctaaataac ccgtatttgt
     2041 gtgtcatgtt tgcatttctg acaagtgaaa caggatctta cgatggagtt ttgtatgaaa
     2101 acaaagttgc agtacgtgac agagtggcat ttgcttgtaa attccttagt gatactcagt
     2161 taaatagata catcgaaaag ttgaccaatg aaatgaaaga ggctggaaat ttggaaggaa
     2221 ttttgcttac aggccttact aaagatggag tggacttaat ggagagttat gttgatagaa
     2281 ctggagatgt tcaaacagca agttactgta tgttacaggg ttcaccttta gatgttctta
     2341 aagatgaaag ggttcagtac tggattgaga attatagaaa tttattagat gcctggaggt
     2401 tttggcataa acgagctgaa tttgatattc acaggagtaa gttggatccc agttccaagc
     2461 ctttagcaca agtttttgtg agttgcaatt tctgtggcaa gtcaatctcc tacagctgtt
     2521 cagctgtgcc tcatcagggc agaggtttta gtcagtatgg tgtgagtggc tcaccaacga
     2581 aatctaaagt cacaagttgt cctggctgtc gaaaaccact tcctcgatgt gcgctttgtc
     2641 tcattaatat gggaacacca gtttctagct gtcctggagg aaccaaatca gatgaaaaag
     2701 tggacttgag caaggacaaa aaattagccc aatttaacaa ctggtttaca tggtgtcata
     2761 attgcaggca cggtggacat gctggacata tgcttagttg gttcagggac catgcagagt
     2821 gccctgtgtc tgcatgcacg tgtaaatgta tgcagttgga tacaacgggg aatctggtac
     2881 ctgcagagac tgtccagcca gcggccgcag gaggtggagg ttacccctat gatgtgcctg
     2941 actacgcatg ataaggatcc ccagcttggc cgccatggcc caacttgttt attgcagctt
     3001 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     3061 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatcggga
     3121 attaattcgg cgcagcacca tggcctgaaa taacctctga aagaggaact tggttaggta
     3181 ccttctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     3241 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     3301 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3361 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     3421 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     3481 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     3541 aaaagctgtt aacagcttgg cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     3601 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     3661 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg
     3721 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
     3781 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     3841 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     3901 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     3961 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
     4021 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     4081 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc gggctattct
     4141 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     4201 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
     4261 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     4321 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     4381 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     4441 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     4501 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     4561 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     4621 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     4681 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     4741 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     4801 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     4861 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     4921 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     4981 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     5041 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     5101 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     5161 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     5221 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     5281 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     5341 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     5401 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     5461 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     5521 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     5581 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     5641 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     5701 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     5761 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     5821 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     5881 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     5941 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     6001 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     6061 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     6121 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     6181 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     6241 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     6301 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     6361 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     6421 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     6481 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     6541 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     6601 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgacatgatt
     6661 acgaattaat tcgagctcgc ccgacattga ttattgacta gttattaata gtaatcaatt
     6721 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
     6781 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
     6841 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     6901 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     6961 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
     7021 acttggcagt acatctacgt attagtcatc gctattacca tg
//
