LOCUS       sequence-177200-        5774 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FLAG-tagged overexpression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5774)
  AUTHORS   Zoncu R, Bar-Peled L, Efeyan A, Wang S, Sancak Y, Sabatini DM
  TITLE     mTORC1 senses lysosomal amino acids through an inside-out mechanism 
            that requires the vacuolar H-ATPase.
  JOURNAL   Science. 2011 Nov 4;334(6056):678-83.
  PUBMED    22053050
REFERENCE   2  (bases 1 to 5774)
  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"; journalName: "Science."; 
            date: "2011-11-4"; volume: "334(6056)"; pages: "678-83"
COMMENT     Sequence Label: pRK5-FLAG-ATP6V0D1
FEATURES             Location/Qualifiers
     source          1..5774
                     /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"
     CDS             937..960
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      2074..2083
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2087..2221
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2124..2143)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2178..2197
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2290..2647
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2290..2481
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2313..2333)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2498..2633
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2667..2684)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2667..2683)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2676..2698)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2896..3351
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2983..3002)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3193..3214
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3368..3387)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3487..3509
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3547..3565)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3633..3737
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3738..3806,3807..4598)
                     /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(3956..3975)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4769..5357
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5258..5277
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5511..5528
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5645..5666
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5681..5686,5687..5704,5705..5711)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5719..5735
                     /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     5724..5746
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5743..5759
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5743..5759
                     /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 attcaccatg gaaatggact acaaggatga cgatgacaag
      961 ggtggtggtg gtggtgcgtc gacgatgtcg ttcttcccgg agctttactt taacgtggac
     1021 aatggctact tggagggact ggtgcgcggc ctgaaggccg gggtgctcag ccaggccgac
     1081 tacctcaacc tggtgcagtg cgagacgcta gaggacttga aactgcatct gcagagcact
     1141 gattatggta acttcctggc caacgaggca tcacctctga cggtgtcagt catcgatgac
     1201 cggctcaagg agaagatggt ggtggagttc cgccacatga ggaaccatgc ctatgagcca
     1261 ctcgccagct tcctagactt cattacttac agttacatga tcgacaacgt gatcctgctc
     1321 atcacaggca cgctgcacca gcgctccatc gctgagctcg tgcccaagtg ccacccacta
     1381 ggcagcttcg agcagatgga ggccgtgaac attgctcaga cacctgctga gctctacaat
     1441 gccattctgg tggacacgcc tcttgcggct tttttccagg actgcatttc agagcaggac
     1501 cttgacgaga tgaacatcga gatcatccgc aacaccctct acaaggccta cctggagtcc
     1561 ttctacaagt tctgcaccct actgggcggg actacggctg atgccatgtg ccccatcctg
     1621 gagtttgaag cagaccgccg cgccttcatc atcaccatca attctttcgg cacagagctg
     1681 tccaaagagg accgtgccaa gctctttcca cactgtgggc ggctctaccc tgagggcctg
     1741 gcgcagctgg ctcgggctga cgactatgaa caggtcaaga acgtggccga ttactacccg
     1801 gagtacaagc tgctcttcga gggtgcaggt agcaaccctg gagacaagac gctggaggac
     1861 cgattctttg agcacgaggt aaagctgaac aagttggcct tcctgaacca gttccacttt
     1921 ggtgtcttct atgccttcgt gaagctcaag gagcaggagt gtcgcaacat cgtgtggatc
     1981 gctgaatgta tcgcccagcg ccaccgcgcc aaaatcgaca actacatccc tatcttctag
     2041 gcggccgcta agtaagtaag gatccccagc ttggccgcca tggcccaact tgtttattgc
     2101 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     2161 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat
     2221 cgggaattaa ttcggcgcag caccatggcc tgaaataacc tctgaaagag gaacttggtt
     2281 aggtaccttc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa
     2341 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     2401 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     2461 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     2521 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     2581 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     2641 ttgcaaaaag ctgttaacag cttggcactg gccgtcgttt tacaacgtcg tgactgggaa
     2701 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     2761 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     2821 tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
     2881 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2941 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     3001 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3061 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     3121 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     3181 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcgggct
     3241 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     3301 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     3361 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     3421 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     3481 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     3541 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     3601 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     3661 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     3721 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     3781 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     3841 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     3901 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     3961 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     4021 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     4081 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     4141 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     4201 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     4261 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     4321 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     4381 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     4441 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     4501 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     4561 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     4621 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     4681 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     4741 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     4801 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     4861 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     4921 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     4981 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     5041 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     5101 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     5161 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     5221 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     5281 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     5341 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     5401 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     5461 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     5521 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     5581 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     5641 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
     5701 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgaca
     5761 tgattacgaa ttaa
//
