LOCUS       sequence-392074-        6193 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Transient expression of Metap2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6193)
  AUTHORS   Shin HR, Citron YR, Wang L, Tribouillard L, Goul CS, Stipp R, 
            Sugasawa Y, Jain A, Samson N, Lim CY, Davis OB, Castaneda-Carpio D, 
            Qian M, Nomura DK, Perera RM, Park E, Covey DF, Laplante M, Evers 
            AS, Zoncu R
  TITLE     Lysosomal GPCR-like protein LYCHOS signals cholesterol sufficiency 
            to mTORC1.
  JOURNAL   Science. 2022 Sep 16;377(6612):1290-1298. doi: 
            10.1126/science.abg6621. Epub 2022 Aug 25.
  PUBMED    36007018
REFERENCE   2  (bases 1 to 6193)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1126/science.abg6621"; journalName: "Science"; date: 
            "2022-09-16- 16"; volume: "377"; issue: "6612"; pages: "1290-1298"
COMMENT     Sequence Label: pRK5-myc Metap2
FEATURES             Location/Qualifiers
     source          1..6193
                     /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             972..1001
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     regulatory      2493..2502
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2506..2640
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2543..2562)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2597..2616
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2709..3066
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2709..2900
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2732..2752)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2917..3052
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3086..3103)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3086..3102)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3095..3117)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3315..3770
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3402..3421)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3612..3633
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3787..3806)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3906..3928
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3966..3984)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4052..4156
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4157..4225,4226..5017)
                     /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(4375..4394)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5188..5776
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5677..5696
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5930..5947
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6064..6085
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6100..6105,6106..6123,6124..6130)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6138..6154
                     /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     6143..6165
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6162..6178
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6162..6178
                     /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 ggagcaaaag ctcatttctg aggaagatct caatggtggt ggtggtgggt
     1021 cgacgatggc gggtgtggag gaggtagcgg cctccgggag ccacctgaat ggcgacctgg
     1081 atccagacga cagggaagaa ggagctgcct ctacggctga ggaagcagcc aagaaaaaaa
     1141 gacgaaagaa gaagaagagc aaagggcctt ctgcagggga acaggaacct gataaagaat
     1201 caggagcctc agtggatgaa gtagcaagac agttggaaag atcagcattg gaagataaag
     1261 aaagagatga agatgatgaa gatggagatg gcgatggaga tggagcaact ggaaagaaga
     1321 agaaaaagaa gaagaagaag agaggaccaa aagttcaaac agaccctccc tcagttccaa
     1381 tatgtgacct gtatcctaat ggtgtatttc ccaaaggaca agaatgcgaa tacccaccca
     1441 cacaagatgg gcgaacagct gcttggagaa ctacaagtga agaaaagaaa gcattagatc
     1501 aggcaagtga agagatttgg aatgattttc gagaagctgc agaagcacat cgacaagtta
     1561 gaaaatacgt aatgagctgg atcaagcctg ggatgacaat gatagaaatc tgtgaaaagt
     1621 tggaagactg ttcacgcaag ttaataaaag agaatggatt aaatgcaggc ctggcatttc
     1681 ctactggatg ttctctcaat aattgtgctg cccattatac tcccaatgcc ggtgacacaa
     1741 cagtattaca gtatgatgac atctgtaaaa tagactttgg aacacatata agtggtagga
     1801 ttattgactg tgcttttact gtcactttta atcccaaata tgatacgtta ttaaaagctg
     1861 taaaagatgc tactaacact ggaataaagt gtgctggaat tgatgttcgt ctgtgtgatg
     1921 ttggtgaggc catccaagaa gttatggagt cctatgaagt tgaaatagat gggaagacat
     1981 atcaagtgaa accaatccgt aatctaaatg gacattcaat tgggcaatat agaatacatg
     2041 ctggaaaaac agtgccgatt gtgaaaggag gggaggcaac aagaatggag gaaggagaag
     2101 tatatgcaat tgaaaccttt ggtagtacag gaaaaggtgt tgttcatgat gatatggaat
     2161 gttcacatta catgaaaaat tttgatgttg gacatgtgcc aataaggctt ccaagaacaa
     2221 aacacttgtt aaatgtcatc aatgaaaact ttggaaccct tgccttctgc cgcagatggc
     2281 tggatcgctt gggagaaagt aaatacttga tggctctgaa gaatctgtgt gacttgggca
     2341 ttgtagatcc atatccacca ttatgtgaca ttaaaggatc atatacagcg caatttgaac
     2401 ataccatcct gttgcgtcca acatgtaaag aagttgtcag caggggagat gactattaag
     2461 cggccgctaa gtaagtaagg atccccagct tggccgccat ggcccaactt gtttattgca
     2521 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     2581 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc
     2641 gggaattaat tcggcgcagc accatggcct gaaataacct ctgaaagagg aacttggtta
     2701 ggtaccttct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag
     2761 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     2821 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     2881 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     2941 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     3001 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     3061 tgcaaaaagc tgttaacagc ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     3121 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     3181 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     3241 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc
     3301 aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     3361 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     3421 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     3481 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg
     3541 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3601 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta
     3661 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     3721 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac
     3781 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     3841 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     3901 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     3961 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     4021 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     4081 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     4141 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     4201 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     4261 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     4321 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     4381 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     4441 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     4501 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     4561 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     4621 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     4681 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     4741 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     4801 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     4861 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     4921 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     4981 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     5041 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     5101 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     5161 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     5221 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     5281 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     5341 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     5401 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     5461 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     5521 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     5581 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     5641 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     5701 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     5761 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     5821 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     5881 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     5941 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     6001 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     6061 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
     6121 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacat
     6181 gattacgaat taa
//
