LOCUS       sequence-174777-        6076 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 6076)
  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 6076)
  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-HA-KPTN
FEATURES             Location/Qualifiers
     source          1..6076
                     /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"
     regulatory      950..959
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             962..988
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      2363..2372
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2376..2510
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2413..2432)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2467..2486
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2579..2936
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2579..2770
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2602..2622)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2787..2922
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2956..2973)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2956..2972)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2965..2987)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3185..3640
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3272..3291)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3482..3503
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3657..3676)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3776..3798
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3836..3854)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3922..4026
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4027..4095,4096..4887)
                     /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(4245..4264)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5058..5646
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5547..5566
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5800..5817
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5934..5955
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5970..5975,5976..5993,5994..6000)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6008..6024
                     /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     6013..6035
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6032..6048
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6032..6048
                     /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 cccgaaccga cagtcggtct cttcaccaag gccattcgcg ccaccatggg
      961 ctacccctat gatgtgcctg actacgcaga tctcaatggt ggtggtggtg ggtcgacgat
     1021 gatgggggag gcggccgtgg ccgcggggcc ttgtccgttg cgcgaggaca gcttcacgcg
     1081 cttctcgtcg cagagcaatg tgtacgggct ggcaggcggc gccggcgggc gcggggagct
     1141 gctggccgcc acccttaaag gcaaggtgct cggcttccgc taccaagacc tccgacagaa
     1201 aatccggcca gtggccaagg agctgcagtt caactacatt cccgtggatg cggagattgt
     1261 ctccatcgac actttcaaca agtcaccccc caagcggggt ctggttgtgg ggatcacgtt
     1321 catcaaggat tcaggggaca agggcagccc cttcctgaac atttactgcg actacgagcc
     1381 cggctctgag tacaaccttg actctattgc ccagagctgc ctgaacctgg agctccagtt
     1441 cactccgttc cagctgtgcc atgcggaggt ccaggtcggg gatcaacttg agactgtgtt
     1501 tctcttgagt gggaacgacc cggccattca tctctacaag gagaacgagg ggctgcatca
     1561 gtttgaggaa cagcccgtgg aaaacctctt cccagagctg acgaacctga ccagtagcgt
     1621 cctctggctg gacgtccaca acttccccgg cacgtcccgg cgcctctcag ctctgggctg
     1681 tcagagtggt tatgtccgtg tcgcccacgt ggaccagcgg agtcgagagg ttctgcagat
     1741 gtggtcggtc ctgcaggacg gtcccatctc ccgagtgatt gtgttcagcc tctcggccgc
     1801 caaggagacc aaggacaggc cactacaaga tgagtacagc gtgctcgtgg ccagcatgtt
     1861 ggagccagca gtggtgtatc gggacctgct gaaccggggt cttgaagacc agcttctcct
     1921 gcccggcagt gaccagtttg acagcgtcct ctgcagcctg gtcaccgatg tggatttgga
     1981 tgggcggcca gaagtcctgg tggccaccta tggacaggaa ctgctgtgtt ataagtaccg
     2041 gggcccagag tcggggcttc ctgaggccca gcacgggttc catctgctgt ggcagcggag
     2101 cttctccagt cccctgctgg ccatggctca cgtggacctg accggggatg ggctgcagga
     2161 gcttgccgtg gtctccctga agggcgtgca catcctgcag cacagcctga ttcaggcctc
     2221 agagctggtc ttgacccggc ttcgacatca agtggagcag aggagacgtc ggctacaggg
     2281 gttggaggac ggggcaggtg cagggcctgc tgagaatgca gcctcttaag cggccgctaa
     2341 gtaagtaagg atccccagct tggccgccat ggcccaactt gtttattgca gcttataatg
     2401 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     2461 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc gggaattaat
     2521 tcggcgcagc accatggcct gaaataacct ctgaaagagg aacttggtta ggtaccttct
     2581 gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     2641 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     2701 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     2761 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     2821 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     2881 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     2941 tgttaacagc ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     3001 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     3061 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat
     3121 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca
     3181 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     3241 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     3301 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     3361 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
     3421 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     3481 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta ttcttttgat
     3541 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     3601 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca
     3661 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     3721 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     3781 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     3841 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     3901 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     3961 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     4021 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     4081 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     4141 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     4201 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     4261 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     4321 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     4381 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     4441 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     4501 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     4561 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     4621 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     4681 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     4741 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     4801 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     4861 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     4921 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     4981 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     5041 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     5101 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     5161 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     5221 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     5281 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     5341 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     5401 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     5461 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     5521 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     5581 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     5641 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     5701 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     5761 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     5821 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     5881 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     5941 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     6001 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacat gattacgaat
     6061 taattcgagc tcgccc
//
