LOCUS       sequence-174774-        8126 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 8126)
  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 8126)
  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 2241-end
FEATURES             Location/Qualifiers
     source          1..8126
                     /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      4413..4422
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    4426..4560
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4463..4482)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4517..4536
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        4629..4986
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4629..4820
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4652..4672)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4837..4972
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5006..5023)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5006..5022)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5015..5037)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5235..5690
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5322..5341)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5532..5553
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5707..5726)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5826..5848
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5886..5904)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5972..6076
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6077..6145,6146..6937)
                     /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(6295..6314)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7108..7696
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7597..7616
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7850..7867
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7984..8005
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8020..8025,8026..8043,8044..8050)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8058..8074
                     /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     8063..8085
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8082..8098
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8082..8098
                     /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 gactctagcc tttgtggatg aaggaggggc ccccttgtca ctggcgttgt
     1021 ggcccccctc ctctccgggg cccccagacc cactgcgaga ggaggaattt gagcaactga
     1081 cccaggtcat ccgctgcccg gttgttgtgg acagttcttc agctcagaat ggggccccac
     1141 ggcttcgatt ggatgtgtgg gaaaagggga acattagtat tgtgcagctg gaggagaaac
     1201 tccgaggagc agctcgccag gccctggccg atgccatcat cgagcttcag ctgctgccag
     1261 cttcactatg tacagaggac acacccacag gaagtctcag gaacggatcg ttggaaacta
     1321 agagctctgc aggccgagct agcacctttc cccctgcccc tgtccctggg gagcctgtga
     1381 ctccacccag caaagcgggc cggcgtagct tctgggatat gctgagtaaa acagaatgtg
     1441 gggatttggg ttcccccaaa acaactgatg acattgtcct ggatcggcca gaagacactc
     1501 ggggccggag gcgtcacaaa accgagagtg ttcggactcc tggtggagct gagcgggcgc
     1561 caggctcaga ttctggagcc cagagacaaa agcgccggac aacacagcta gaagagggtg
     1621 aggtggggac ccttcatcct gtgtttgccc gtgttgctca gcgctggatg gagtttatgg
     1681 ttcagattgg ttgtgcctca gtgtccagaa gctctgccca catggtgtcc cggttcctcc
     1741 ttccatccat cctgtctgag ttcaccgcac tggtcacctc aatggctgga gacaccagtg
     1801 tccgcatctt tgagcagcat ttgggttcag agccagagat cttcggccct tgttcccctg
     1861 ggcaactggg cccctctccc cgccctgcag ctgagcggca tctgctgctt ctgggaagga
     1921 acttcttgca gtggaggaga ccaacacagc aggctgccaa agccatgcag cgcttcgagc
     1981 caggaggtga tgggagctca gggcgaaatg ctccccggca gaggctcttg ctactagagg
     2041 ttgtggacaa gaagctacag ctgctgacct acaactgggc tccagacctg ggggcagcat
     2101 tgggccgagc gctggttcgc ctggtgcagt ggcagaatgc acgagcccat ctcatcttct
     2161 gcctactcag ccagaagctt ggcctcttcc atcattatgg ccagttggac ttccccgtgc
     2221 gagatgaaaa ggagccaaac ccattcctgc tgccgaccat ggaagtggag accctcatcc
     2281 ggagtgcaag tcccccgctg agccgtgagc agggccgact gagtgggtcc tctcgtggtg
     2341 ggggtcctct tcccctggac acattcccct ttgacgaggc cctaagggat atcacggctg
     2401 cccgccccag ctccgtactt ggtcctgtgc ccagacctcc tgatcctgtc acctaccatg
     2461 gacaacagtt cctagagatc aagatggcag agcgcagaga gctggagcgc cagatgaaga
     2521 tggaaaacct gtttgtaacc tggcagcagc gttctacccc agccaccatg cccatcagtg
     2581 ctggagagct ggagaccctg aagcagtcat cccgcctggt gcattactgt gcaacagcca
     2641 tgctcttcga cccagctgcc tggctgcatg ggcccccaga gacctctgga ccccctgacg
     2701 ggcagcggcg ccatcgccct gagtcagggt ctgggagccg agaggccccc acaagctgtg
     2761 aatccttgga tgtgtcgccc ccgggagccc gtgaggagcc ttggctgaag gagctgagct
     2821 tggctttcct gcagcaatat gtgcagtatc tgcagagcat aggttttgtg ctggtaccac
     2881 tgcggccccc ctcacccgcc cgcagcacca gccggccacg ggccatggct atccttggaa
     2941 cagagggtcg aggctccttc tcctgcccta aaaccaagac tgatgggagc cccaagagca
     3001 ctagctctcc ggtaaccacc taccacctgc agcgggcact gcctgggggc atcatcctca
     3061 tggaactggc attccagggc tgttacttct gtgtcaaaca gtttgccctg gaatgttccc
     3121 gaatcccaat ggggcaggct gtcaactcac agctgtccat gctgttcaca gaggagtgtg
     3181 acaaggtgcg ggacctgatg cacgtgcact cgttcagcta tgacttccat ctgcgcctcg
     3241 tgcatcagca cgtgctaggt gcccatctgg tgctgcggca cggctaccac ctcaccacct
     3301 ttctgcgaca cttcctggcc caccaccctg acggacccca ctttggccgc aatcacattt
     3361 accaagggac attggagctc cccacaccac tcattgctgc ccaccagcta tacaactacg
     3421 tggctgatca cgccagctct taccacatga agccattgcg aatggcccgg ccagggggcc
     3481 cagaacacaa cgagtatgcc ctggtgtcgg catggcacag ttctggctcc tacctggact
     3541 ctgagggact tcgacaccag gatgactttg atgtgtctct gcttgtctgt cactgtgctg
     3601 caccctttga ggagcaagga gaggctgagc ggcacgttct gcggctacag ttcttcgtgg
     3661 tgctcaccag ccagcgagag ctcttcccca ggctcactgc tgacatgcgc cgcttccgga
     3721 agccacccag actgccccct gagccagagg ctcctgggag ttcagctggc agccctgggg
     3781 aggcctcagg gcttattcta gcgcctggac cggctcctct gttcccacca ctggctgcag
     3841 aggtgggcat ggcacgagca cggctggctc agctggtgcg gctggctgga gggcactgcc
     3901 gtcgggacac cctttggaag cgcctcttct tgctggagcc accggggcct gatcgactgc
     3961 ggctaggggg gcgcctggcc ctggcagagc tggaggaact cctagaagca gtccatgcca
     4021 aatccattgg ggacatcgac ccccagctgg actgcttcct atccatgacg gtctcctggt
     4081 accagagcct gatcaaagtt ctcctaagcc gcttccccca gagctgtcgc catttccaaa
     4141 gcccagactt gggaactcag tacctggttg tgctgaatca gaagttcact gactgctttg
     4201 tgctagtgtt tctggactcc cacttaggaa agacgtctct gacagtggtt ttccgagagc
     4261 ccttcccagt acagccccag gacagcgaga gcccccctgc ccaactggtc tccacctacc
     4321 accacctgga gtctgtcatc aacacagcct gtttcaccct ctggacccgc ctcctctgag
     4381 cggccgctaa gtaagtaagg atccccagct tggccgccat ggcccaactt gtttattgca
     4441 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     4501 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc
     4561 gggaattaat tcggcgcagc accatggcct gaaataacct ctgaaagagg aacttggtta
     4621 ggtaccttct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag
     4681 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     4741 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     4801 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     4861 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     4921 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     4981 tgcaaaaagc tgttaacagc ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     5041 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     5101 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     5161 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc
     5221 aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     5281 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     5341 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     5401 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg
     5461 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     5521 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta
     5581 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     5641 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac
     5701 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     5761 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     5821 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     5881 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     5941 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     6001 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     6061 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     6121 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     6181 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     6241 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     6301 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     6361 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     6421 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     6481 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     6541 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     6601 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     6661 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     6721 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     6781 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     6841 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     6901 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     6961 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     7021 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     7081 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     7141 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     7201 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     7261 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     7321 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     7381 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     7441 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     7501 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     7561 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     7621 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     7681 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     7741 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     7801 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     7861 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     7921 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     7981 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
     8041 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacat
     8101 gattacgaat taattcgagc tcgccc
//
