LOCUS       sequence-174764-        7781 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of SZT2 fragment.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7781)
  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 7781)
  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-SZT2 1-1021
FEATURES             Location/Qualifiers
     source          1..7781
                     /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      4068..4077
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    4081..4215
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4118..4137)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4172..4191
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        4284..4641
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4284..4475
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4307..4327)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4492..4627
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4661..4678)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4661..4677)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4670..4692)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4890..5345
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4977..4996)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5187..5208
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5362..5381)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5481..5503
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5541..5559)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5627..5731
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5732..5800,5801..6592)
                     /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(5950..5969)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6763..7351
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7252..7271
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7505..7522
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7639..7660
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7675..7680,7681..7698,7699..7705)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7713..7729
                     /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     7718..7740
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7737..7753
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7737..7753
                     /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 gatggcctcg gagcgcccgg agccggaggt ggaagaagct gggcaggtgt
     1021 tcctgttaat gaaaaaggat tatcgaatct cccgaaatgt tcgcctggct tggttcctca
     1081 gtcatctgca ccaaactgtg caggccacac cccaggagat gctgcttcag tctgaacagg
     1141 aattggaagt cctcagtgtc ctgccccctg ggtggcagcc agatgaacca gtggtcccaa
     1201 ggccattcct cctggtacct tccacccggg tcaccttcct ggcttggcag tatcggtttg
     1261 tcattgagtt ggaccttagc ccatctactg gcattgtgga tgattccaca ggggagatct
     1321 tgtttgatga agttttccat gccctgtccc gctgcttagg cgggctgctt cggcccttcc
     1381 gagtgcctgg atcttgcatc gacttccagc ctgagatcta tgtaactatc caggcctact
     1441 cctccatcat tggactgcag tcccaccagg tgctggtaca gggctgcctc ttggaccctt
     1501 cccagcggga ggtgttcctg cagcagatat atgagcagct ctgcctcttt gaggataagg
     1561 tggccaccat gctgcagcag cagtacgatc cccagagcca ggcagaagac cagtccccag
     1621 actcagggga cctactgggc cggaaggtag gcgtctccat ggtgacagct gatcttgggc
     1681 tggtcagtat gattcgtcag ggcatcttgg cactgcagtt actaccctcg aactctagtg
     1741 cagggattat cgtgatcacg gatggggtga ccagtgtacc tgatgttgct gtctgtgaga
     1801 cactgctgaa ccagcttcgc agtggcactg tggcttgttc ctttgtccag gtgggaggag
     1861 tttactctta tgactgcagt tttggccatg tgcccaatgt ggaattaatg aagttcatcg
     1921 caatggcaac atttgggtcc tacctgtcca cttgtcctga gccggagcca ggcaacctgg
     1981 gtctgactgt ctaccaccgg gcatttctcc tctattcctt cctgcgcagt ggggaagcac
     2041 tgaaccctga atattactgc ggctctcagc accgcctatt taatgagcac ctggtctctg
     2101 caagcagcaa ccctgccctg gccttgcgcc ggaagaagca cactgagaag gaggtgccag
     2161 ccgacttggt cagcactgtg tccgtacggc ttcgagaggg ctacagtgtc cgagaggtca
     2221 cactggccaa aggagggtcc caattggagg taaagctggt gctgctgtgg aaacacaaca
     2281 tgcgcattga gtatgtggct atggcaccct ggcccctgga gcctgagggc cctcgagtaa
     2341 cacgggtgga agtgacgatg gaaggcggct acgacatttt gcatgatgtg tcctgtgcac
     2401 taaggcagcc cattcgttca ttgtatcgta cccatgttat ccggcgtttc tggaacacgc
     2461 tgcagagcat caaccagaca gaccagatgc ttgcccacct tcagtccttc tcctcagtgc
     2521 ctgagcattt cacgcttcct gacagcacca agagcggagt gccactcttc tacatccctc
     2581 caggctccac caccccggtg ctctccctcc agcccagtgg ttctgactca tcccatgccc
     2641 agtttgctgc ctactggaag ccagtgctgt ccatggatgc aaattcctgg cagcgatggc
     2701 tgcacatgca tcgcctggtg ctaatcctgg agcatgacac accaatcccc aagcacttgc
     2761 acaccccggg cagcaatggg cgctacagca ctatccagtg caggatctcc cactcctccc
     2821 tgacctctct gctgcgggac tggagcagct tcgtactagt cgagggctat tcttatgtta
     2881 agctgctctc cagtgcccca gaccagcccc ccaattcctt ctacatggtc cgtatcattt
     2941 ccaaggcccc atgcatggtt cttcgcctgg gttttcccat tggcacacca gcaccggccc
     3001 ggcacaagat tgtgtcaggc ttgagggaag agatcctgcg gctgcgtttc ccccaccggg
     3061 tacaaagcaa ggagccaacg cccaaggtga aacgaaaagg gctagggggt gctggtgggg
     3121 gcagctctcc ctccaagtca ccccccgtgc tggggccaca gcaggccctg tctgaccggc
     3181 cctgccttgt ggtcctgcat aagccactgg acaaactgct catcaggtat gagaagctgc
     3241 ccttggacta ccgggcaccc ttcttgctga cattggagcc accaggtcca ctgcccttgg
     3301 tgtcaggccg ctcagcctct tctagcctgg cgtcactgtc ccgctacctc taccatcagc
     3361 gctggctttg gagtgtcccg tcaggactgg cccctgcgct gcctctcagt gccattgccc
     3421 agctcctctc catcctcact gaagtccgac tttctgaagg attccacttc gcctgcagtg
     3481 gggaaggaat catcaacatg gttctggagc ttccaattca gaatgaacca ccagggcagg
     3541 ctgcagctga agagaagcac acctgtgttg tccagtacat cctcttcccc ccacactcta
     3601 cctccaccaa agacagcttc tcgacagatg atgacaatga tgtggaagtg gaggccctgg
     3661 agggagactc agagctcaat ctggtcactg aggtgtgggt ggagccacag tatgggcgag
     3721 tgggacctgg ccctggaatc tggaagcacc tccaggacct gacgtattct gagatcccgc
     3781 aagctctcca cccacgggat gctgcctgca taggctccat gctgagcttt gaatacctga
     3841 tacagctgtg tcagagcaag gaatggggtc ctctgccccc agagccgagg gtctctgatg
     3901 gattggatca gggaggagac acctgcgtcc atgagatccc tttccatttt gacctaatgg
     3961 gattgctgcc acagtgccag cagctccaga tgttcttcct cttgcttgcc agaggtgccg
     4021 aggggccact gtgagcggcc gctaagtaag taaggatccc cagcttggcc gccatggccc
     4081 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     4141 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     4201 tatcatgtct ggatcgggaa ttaattcggc gcagcaccat ggcctgaaat aacctctgaa
     4261 agaggaactt ggttaggtac cttctgaggc ggaaagaacc agctgtggaa tgtgtgtcag
     4321 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     4381 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     4441 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     4501 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     4561 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     4621 ggaggcctag gcttttgcaa aaagctgtta acagcttggc actggccgtc gttttacaac
     4681 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     4741 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     4801 gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     4861 cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc
     4921 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     4981 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     5041 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     5101 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     5161 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     5221 ccctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     5281 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
     5341 aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     5401 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     5461 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     5521 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     5581 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     5641 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     5701 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     5761 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     5821 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     5881 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     5941 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     6001 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     6061 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     6121 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     6181 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     6241 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     6301 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     6361 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     6421 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     6481 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     6541 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     6601 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     6661 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     6721 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     6781 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     6841 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     6901 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     6961 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     7021 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     7081 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     7141 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     7201 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     7261 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     7321 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     7381 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     7441 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     7501 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
     7561 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     7621 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
     7681 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
     7741 aaacagctat gacatgatta cgaattaatt cgagctcgcc c
//
