LOCUS       sequence-400874-        9055 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of VPS15 with TwinStrep-Flag tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9055)
  AUTHORS   Chen M, Ren X, Nguyen TN, Khuu G, Cook ASI, Lazarou M, Hurley JH
  TITLE     Structure and activation of the human autophagy-initiating 
            ULK1C:PI3KC3-C1 supercomplex
  JOURNAL   bioRxiv 2023.06.01.543278
REFERENCE   2  (bases 1 to 9055)
  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: "bioRxiv 
            2023.06.01.543278"
COMMENT     Sequence Label: pCAG-VPS15-TSF
FEATURES             Location/Qualifiers
     source          1..9055
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        132..511
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        513..788
                     /label=chicken beta-actin promoter
     intron          789..1805
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1729..1751
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1813..1832
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1879..1888
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5965..5985
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             5998..6021
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             6058..6081
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             6088..6111
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(6204..6223)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    6269..6324
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(6323..6342)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(6685..6701)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6685..6701)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6698..6720)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6709..6725
                     /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)."
     promoter        complement(join(6733..6739,6740..6757,6758..6763))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6778..6799
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6858..7053
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6904..7039
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    7059..7193
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7096..7115)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7150..7169
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7261..7278)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7432..8020)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7512..7531)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8191..8982,8983..9051))
                     /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     8814..8833
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(9052..9055,1..101))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
       61 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
      121 ccacctgggt cgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
      181 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
      241 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
      301 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
      361 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
      421 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
      481 catctacgta ttagtcatcg ctattaccat ggtcgaggtg agccccacgt tctgcttcac
      541 tctccccatc tcccccccct ccccaccccc aattttgtat ttatttattt tttaattatt
      601 ttgtgcagcg atgggggcgg gggggggggg ggsgcgcgcc aggcggggcg gggcggggcg
      661 aggggcgggg cggggcgagg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc
      721 gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc
      781 ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc
      841 gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc
      901 cttctcctcc gggctgtaat tagcgcttgg tttaatgacg gcttgtttct tttctgtggc
      961 tgcgtgaaag ccttgagggg ctccgggagg gccctttgtg cggggggagc ggctcggggg
     1021 gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg ctccgcgctg cccggcggct
     1081 gtgagcgctg cgggcgcggc gcggggcttt gtgcgctccg cagtgtgcgc gaggggagcg
     1141 cggccggggg cggtgccccg cggtgcgggg ggggctgcga ggggaacaaa ggctgcgtgc
     1201 ggggtgtgtg cgtggggggg tgagcagggg gtgtgggcgc gtcggtcggg ctgcaacccc
     1261 ccctgcaccc ccctccccga gttgctgagc acggcccggc ttcgggtgcg gggctccgta
     1321 cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg ggggtgccgg
     1381 gcggggcggg gccgcctcgg gccggggagg gctcggggga ggggcgcggc ggcccccgga
     1441 gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg taatcgtgcg
     1501 agagggcgca gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct gggaggcgcc
     1561 gccgcacccc ctctagcggg cgcggggcga agcggtgcgg cgccggcagg aaggaaatgg
     1621 gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct tctccctctc cagcctcggg
     1681 gctgtccgcg gggggacggc tgccttcggg ggggacgggg cagggcgggg ttcggcttct
     1741 ggcgtgtgac cggcggctct agagcctctg ctaaccatgt tcatgccttc ttctttttcc
     1801 tacagctcct gggcaacgtg ctggttattg tgctgtctca tcattttggc aaagaattcc
     1861 tcgatcgacg gtatcgaagc caccatggga aatcagctgg ccggcatcgc tcctagccag
     1921 attctgtccg tggaatctta cttcagtgac atccatgatt ttgagtatga caaatcactg
     1981 ggaagcaccc ggttctttaa ggtggcccgg gctaaacaca gagaaggcct ggtggtggtg
     2041 aaggtgttcg ctatccagga tcccactctg cctctgacca gttataaaca ggagctggag
     2101 gaactgaaga ttagactgaa ctcagcccag aattgcctgc cattccagaa agctagcgag
     2161 aaggcctccg aaaaagccgc tatgctgttt aggcagtacg tgcgcgacaa cctgtatgat
     2221 cggatttcca caagaccctt cctgaacaat atcgagaagc ggtggatcgc ctttcagatt
     2281 ctgactgctg tggaccaggc ccataaatct ggagtgagac acggcgatat caagactgag
     2341 aacgtgatgg tgaccagctg gaattgggtg ctgctgacag acttcgcttc ctttaagcca
     2401 acttacctgc ccgaagacaa ccctgccgat ttcaattatt tctttgacac tagccggaga
     2461 aggacctgct acatcgctcc tgagcggttc gtggatggcg ggatgtttgc caccgagctg
     2521 gaatacatga gagacccatc aacacccctg gtggatctga acagcaatca gaggaggaga
     2581 ggagagctga agagggctat ggacatcttt tctgctgggt gtgtgattgc cgagctgttc
     2641 accgaaggag tgcctctgtt tgatctgagt cagctgctgg cctataggaa cggacacttc
     2701 tttccagagc aggtgctgaa taagatcgag gatcatagca ttcgcgaact ggtgacacag
     2761 atgatccaca gggaaccaga caaacgcctg gaggctgaag attacctgaa gcagcagaga
     2821 ggcaacgcct tcccagagat cttctacaca tttctgcagc cctatatggc ccagttcgct
     2881 aaagagactt ttctgagcgc cgacgaacgg atcctggtca tcagaaagga tctgggaaac
     2941 atcattcata atctgtgcgg ccacgacctg cccgaaaaag ccgagggcga acctaaggag
     3001 aatgggctgg tcatcctggt gtctgtgatt acaagttgcc tgcagactct gaagtattgt
     3061 gatagcaaac tggccgctct ggaactgatc ctgcatctgg ctccaaggct gtccgtggag
     3121 atcctgctgg acagaattac cccttacctg ctgcacttct ccaacgattc tgtgccacgg
     3181 gtgagagccg aggctctgag gacactgact aaagtgctgg ccctggtgaa ggaagtgcct
     3241 cgcaacgaca tcaatatcta ccccgagtat atcctgcctg gcattgccca cctggctcag
     3301 gacgatgcca ctattgtgag gctggcctat gctgagaata tcgccctgct ggctgaaacc
     3361 gccctgcgct ttctggagct ggtgcagctg aagaacctga atatggaaaa cgaccccaac
     3421 aatgaggaaa tcgatgaggt gacccatcct aacgggaatt acgacacaga gctgcaggcc
     3481 ctgcacgaaa tggtgcagca gaaagtggtg accctgctgt ccgatccaga aaacattgtg
     3541 aagcagactc tgatggagaa tgggatcacc aggctgtgtg tgttctttgg acgccagaaa
     3601 gctaacgacg tgctgctgtc acatatgatc acattcctga acgacaagaa tgattggcac
     3661 ctgcggggcg ccttctttga tagcatcgtg ggagtggccg cttatgtggg ctggcagagc
     3721 tcctctattc tgaagcctct gctgcagcag gggctgtctg acgccgagga atttgtgatc
     3781 gtgaaagctc tgtatgccct gacctgcatg tgtcagctgg gactgctgca gaagccacat
     3841 gtgtacgagt tcgctagcga tatcgcccca tttctgtgcc accccaatct gtggattcgg
     3901 tatggggccg tgggattcat cactgtggtg gctagacaga ttagcaccgc cgacgtgtac
     3961 tgtaaactga tgccatacct ggatccctat atcacacagc ccatcattca gattgaacgg
     4021 aaactggtgc tgctgtccgt gctgaaggag cctgtgtcta ggagtatttt cgactacgcc
     4081 ctgcgcagca aggatatcac ttccctgttt agacacctgc atatgaggca gaagaaacgc
     4141 aacggatccc tgcctgactg cccacctcca gaggatccag ccatcgctca gctgctgaag
     4201 aaactgctgt ctcaggggat gaccgaggaa gaggaagaca agctgctggc cctgaaagat
     4261 ttcatgatga agtctaacaa ggccaaagct aatatcgtgg accagagtca cctgcatgat
     4321 agttcacaga agggcgtgat cgacctggcc gctctgggca ttacagggag gcaggtggat
     4381 ctggtgaaga ctaaacagga gcccgacgat aaaagggccc gcaagcacgt gaaacaggac
     4441 agcaacgtga atgaggaatg gaagtcaatg tttggaagcc tggacccccc taatatgcct
     4501 caggctctgc caaaaggcag cgatcaggaa gtgatccaga ccgggaagcc accaagatca
     4561 gagagctccg ccggcatttg tgtgcccctg agcacatcta gtcaggtgcc tgaggtgacc
     4621 acagtgcaga acaagaaacc tgtgatccca gtgctgtcaa gcaccattct gccaagcaca
     4681 taccagatca ggattactac ctgcaaaacc gaactgcagc agctgatcca gcagaagcgg
     4741 gagcagtgta acgctgaaag aattgccaag cagatgatgg agaatgccga gtgggaaagt
     4801 aaacctccac cacctggatg gaggccaaag ggactgctgg tggctcacct gcatgaacac
     4861 aaaagcgccg tgaatcggat cagagtgtcc gacgagcact ctctgttcgc cacatgctcc
     4921 aacgatggaa ctgtgaagat ttggaattct cagaagatgg agggcaaaac aactaccaca
     4981 aggtctatcc tgacctatag tcggattgga ggcagagtga agaccctgac attttgtcag
     5041 ggcagccatt acctggccat cgcttccgac aacggggctg tgcagctgct gggaattgaa
     5101 gcctcaaagc tgcccaaaag ccctaagatc caccccctgc agtccaggat tctggaccag
     5161 aaggaggatg gctgcgtggt ggacatgcac catttcaaca gtggggccca gtcagtgctg
     5221 gcctacgcta cagtgaatgg gtctctggtg ggatgggatc tgcgctcctc tagtaacgct
     5281 tggactctga agcatgacct gaaatccggc ctgatcacct ctttcgccgt ggatattcac
     5341 cagtgctggc tgtgtatcgg cacatcaagc gggactatgg cttgctggga catgcgcttt
     5401 cagctgccaa tctcctctca ttgtcaccca agcagggcta ggattaggag gctgtctatg
     5461 caccctctgt atcagagttg ggtcatcgcc gctgtgcagg ggaacaatga agtgtccatg
     5521 tgggacatgg agaccggaga tcggagattc acactgtggg ctagttcagc cccacccctg
     5581 tctgagctgc agcctagtcc acattcagtg cacgggatct actgttcccc tgctgatgga
     5641 aatccaatcc tgctgaccgc cggctctgac atgaaaatta ggttttggga tctggcttac
     5701 ccagagcgca gttatgtggt ggccggctca accagctccc cctcagtgag ctactatcgg
     5761 aagatcattg agggcacaga agtggtgcag gaaatccaga acaagcagaa agtgggacca
     5821 agtgacgata ctccaaggag gggacctgag tcactgccag tgggacacca tgacatcatt
     5881 accgatgtgg ctacattcca gactacccag ggcttcatcg tgaccgcctc cagagacggc
     5941 atcgtgaaag tgtggaaggg taccgaaaac ctgtacttcc aatccggtat ggcggcctgg
     6001 tcacacccgc agttcgagaa aggtggaggt gcccgaggtg gatcgggagg tggatcgtgg
     6061 agccacccgc agttcgaaaa aggattcgat tacaaggatg acgatgacaa gtagctcgag
     6121 gaattcactc ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc
     6181 ctggctcaca aataccactg agatcttttt ccctctgcca aaaattatgg ggacatcatg
     6241 aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg
     6301 tgttggaatt ttttgtgtct ctcactcgga aggacatatg ggagggcaaa tcatttaaaa
     6361 catcagaatg agtatttggt ttagagtttg gcaacatatg cccatatgct ggctgccatg
     6421 aacaaaggtt ggctataaag aggtcatcag tatatgaaac agccccctgc tgtccattcc
     6481 ttattccata gaaaagcctt gacttgaggt tagatttttt ttatattttg ttttgtgtta
     6541 tttttttctt taacatccct aaaattttcc ttacatgttt tactagccag atttttcctc
     6601 ctctcctgac tactcccagt catagctgtc cctcttctct tatggagatc cctcgacctg
     6661 cagcccaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     6721 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     6781 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     6841 gtcgtgccag cggatccgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     6901 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     6961 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     7021 ggaggctttt ttggaggcct aggcttttgc aaaaagctaa cttgtttatt gcagcttata
     7081 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     7141 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg atccgctgca
     7201 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     7261 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     7321 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     7381 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     7441 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     7501 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     7561 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     7621 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     7681 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     7741 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     7801 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     7861 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     7921 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     7981 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     8041 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     8101 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     8161 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     8221 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     8281 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     8341 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     8401 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     8461 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     8521 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     8581 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     8641 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     8701 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     8761 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     8821 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     8881 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     8941 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     9001 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactc
//
