LOCUS       sequence-174776-        6029 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of KPTN.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6029)
  AUTHORS   Wolfson RL, Chantranupong L, Wyant GA, Gu X, Orozco JM, Shen K, 
            Condon KJ, Petri S, Kedir J, Scaria SM, Abu-Remaileh M, Frankel WN, 
            Sabatini DM
  TITLE     KICSTOR recruits GATOR1 to the lysosome and is necessary for 
            nutrients to regulate mTORC1.
  JOURNAL   Nature. 2017 Feb 15. doi: 10.1038/nature21423.
  PUBMED    28199306
REFERENCE   2  (bases 1 to 6029)
  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: "Nature. 
            2017 Feb 15. doi: 10.1038/nature21423."
COMMENT     Sequence Label: pRK5-FLAG-KPTN
FEATURES             Location/Qualifiers
     source          1..6029
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     581..605
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        815..833
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     815..832
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     846..865
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             924..947
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      2316..2325
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2329..2463
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2366..2385)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2420..2439
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2532..2889
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2532..2723
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2555..2575)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2740..2875
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2909..2926)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2909..2925)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2918..2940)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3138..3593
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3225..3244)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3435..3456
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3610..3629)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3729..3751
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3789..3807)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3875..3979
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3980..4048,4049..4840)
                     /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(4198..4217)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5011..5599
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5500..5519
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5753..5770
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5887..5908
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5923..5928,5929..5946,5947..5953)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5961..5977
                     /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     5966..5988
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5985..6001
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5985..6001
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc
      601 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
      661 ccagcctccg cggccgggaa cggtgcattg gaacgcggat tccccgtgcc aagagtgacg
      721 taagtaccgc ctatagagtc tataggccca cccccttggc ttcgttagaa cgcggctaca
      781 attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat agaataacat
      841 ccactttgcc tttctctcca caggtgtcca ctcccaggtc caactgcacc tcggttctat
      901 cgattgaatt caccatggaa atggactaca aggatgacga tgacaagggt ggtggtggtg
      961 gtgcgtcgac gatgatgggg gaggcggccg tggccgcggg gccttgtccg ttgcgcgagg
     1021 acagcttcac gcgcttctcg tcgcagagca atgtgtacgg gctggcaggc ggcgccggcg
     1081 ggcgcgggga gctgctggcc gccaccctta aaggcaaggt gctcggcttc cgctaccaag
     1141 acctccgaca gaaaatccgg ccagtggcca aggagctgca gttcaactac attcccgtgg
     1201 atgcggagat tgtctccatc gacactttca acaagtcacc ccccaagcgg ggtctggttg
     1261 tggggatcac gttcatcaag gattcagggg acaagggcag ccccttcctg aacatttact
     1321 gcgactacga gcccggctct gagtacaacc ttgactctat tgcccagagc tgcctgaacc
     1381 tggagctcca gttcactccg ttccagctgt gccatgcgga ggtccaggtc ggggatcaac
     1441 ttgagactgt gtttctcttg agtgggaacg acccggccat tcatctctac aaggagaacg
     1501 aggggctgca tcagtttgag gaacagcccg tggaaaacct cttcccagag ctgacgaacc
     1561 tgaccagtag cgtcctctgg ctggacgtcc acaacttccc cggcacgtcc cggcgcctct
     1621 cagctctggg ctgtcagagt ggttatgtcc gtgtcgccca cgtggaccag cggagtcgag
     1681 aggttctgca gatgtggtcg gtcctgcagg acggtcccat ctcccgagtg attgtgttca
     1741 gcctctcggc cgccaaggag accaaggaca ggccactaca agatgagtac agcgtgctcg
     1801 tggccagcat gttggagcca gcagtggtgt atcgggacct gctgaaccgg ggtcttgaag
     1861 accagcttct cctgcccggc agtgaccagt ttgacagcgt cctctgcagc ctggtcaccg
     1921 atgtggattt ggatgggcgg ccagaagtcc tggtggccac ctatggacag gaactgctgt
     1981 gttataagta ccggggccca gagtcggggc ttcctgaggc ccagcacggg ttccatctgc
     2041 tgtggcagcg gagcttctcc agtcccctgc tggccatggc tcacgtggac ctgaccgggg
     2101 atgggctgca ggagcttgcc gtggtctccc tgaagggcgt gcacatcctg cagcacagcc
     2161 tgattcaggc ctcagagctg gtcttgaccc ggcttcgaca tcaagtggag cagaggagac
     2221 gtcggctaca ggggttggag gacggggcag gtgcagggcc tgctgagaat gcagcctctt
     2281 aagcggccgc taagtaagta aggatcccca gcttggccgc catggcccaa cttgtttatt
     2341 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     2401 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
     2461 atcgggaatt aattcggcgc agcaccatgg cctgaaataa cctctgaaag aggaacttgg
     2521 ttaggtacct tctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga
     2581 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2641 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2701 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     2761 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     2821 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     2881 ttttgcaaaa agctgttaac agcttggcac tggccgtcgt tttacaacgt cgtgactggg
     2941 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     3001 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     3061 aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     3121 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     3181 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     3241 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3301 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     3361 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     3421 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggg
     3481 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     3541 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
     3601 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     3661 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     3721 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     3781 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     3841 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     3901 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     3961 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     4021 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     4081 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     4141 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     4201 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     4261 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     4321 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     4381 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     4441 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     4501 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     4561 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     4621 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     4681 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     4741 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     4801 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     4861 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     4921 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     4981 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     5041 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     5101 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     5161 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     5221 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     5281 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     5341 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     5401 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     5461 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     5521 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     5581 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     5641 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     5701 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     5761 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     5821 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     5881 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
     5941 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
     6001 catgattacg aattaattcg agctcgccc
//
