LOCUS       sequence-358523-        5650 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector for ULK1 MIT domains, residues 833-1050.
            Has Precision protease cleavable N-terminal One-Strep Flag tag. 
            Internal ID: WISP20-37..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5650)
  AUTHORS   Wenzel DM, Mackay DR, Skalicky JJ, Paine EL, Miller MS, Ullman KS, 
            Sundquist WI
  TITLE     Comprehensive analysis of the human ESCRT-III-MIT domain interactome
            reveals new cofactors for cytokinetic abscission.
  JOURNAL   Elife. 2022 Sep 15;11:e77779. doi: 10.7554/eLife.77779.
  PUBMED    36107470
REFERENCE   2  (bases 1 to 5650)
  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: "Elife."; 
            date: "2022-09-15"; volume: "11:e77779. doi"; pages: " 
            10.7554/eLife.77779"
COMMENT     Sequence Label: pCAG-OSF-PP-ULK1(MIT)2, residues 833-1050
FEATURES             Location/Qualifiers
     source          1..5650
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     gap             503..532
                     /estimated_length=30
     intron          665..1681
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1605..1627
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1689..1708
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1755..1764
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1770..1793
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1830..1853
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1860..1883
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1896..1919
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(2671..2690)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2736..2791
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2790..2809)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3152..3168)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3152..3168)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3165..3187)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3176..3192
                     /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(3200..3206,3207..3224,3225..3230))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3245..3266
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3325..3520
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3371..3506
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3526..3660
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3563..3582)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3617..3636
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3728..3745)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3899..4487)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3979..3998)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4658..5449,5450..5518))
                     /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     5281..5300
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5519..5623)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      421 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      481 cgatgggggc gggggggggg ggnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnggcgaggg
      541 gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa
      601 gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg
      661 ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg
      721 cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc
      781 tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc tgtggctgcg
      841 tgaaagcctt gaggggctcc gggagggccc tttgtgcggg gggagcggct cggggggtgc
      901 gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc gcgctgcccg gcggctgtga
      961 gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg ggagcgcggc
     1021 cgggggcggt gccccgcggt gcgggggggg ctgcgagggg aacaaaggct gcgtgcgggg
     1081 tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc aaccccccct
     1141 gcacccccct ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc tccgtacggg
     1201 gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg
     1261 ggcggggccg cctcgggccg gggagggctc gggggagggg cgcggcggcc cccggagcgc
     1321 cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat cgtgcgagag
     1381 ggcgcaggga cttcctttgt cccaaatctg tgcggagccg aaatctggga ggcgccgccg
     1441 caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg
     1501 ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc ctcggggctg
     1561 tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg
     1621 tgtgaccggc ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca
     1681 gctcctgggc aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattcctcga
     1741 tcgacggtat cgatgccacc atggctagct ggagccaccc gcagttcgag aaaggtggag
     1801 gtgcccgagg tggatcggga ggtggatcgt ggagccaccc gcagttcgaa aaaggattcg
     1861 attacaagga tgacgatgac aagggcacaa gatctctgga agtcctgttc cagggcccgg
     1921 gtaccgagac cctcatggag caagagcaca cggagatcct gcgtggcctg cgcttcacgc
     1981 tgctgttcgt gcagcacgtc ctggagatcg cagccctgaa gggcagcgcc agtgaggcgg
     2041 cggggggccc tgagtaccag ctgcaggaga gtgtggtggc cgaccagatc agcctgctga
     2101 gccgagaatg gggcttcgcg gaacagctgg tgctgtacct gaaggtggcc gagctactgt
     2161 cctccggcct gcaaagtgcc atcgaccaga tccgggccgg caagctctgc ctgtcgtcca
     2221 ctgtgaagca ggtggtgcgc aggctgaatg agctgtacaa ggccagcgtg gtgtcctgcc
     2281 agggcctgag cctgcggctg cagcgcttct tcctggacaa gcagcggctc ctggaccgca
     2341 ttcacagcat cactgccgag aggctcatct tcagccacgc tgtgcagatg gtgcagtcgg
     2401 ctgccctgga cgagatgttc cagcaccgtg agggctgcgt cccacgctac cacaaggccc
     2461 tgctgctcct ggaggggctg cagcacatgc tctcggacca ggccgacatc gagaacgtca
     2521 ccaagtgcaa gctgtgcatt gagcggagac tctcggcgct gctgactggc atctgtgcct
     2581 gatcgaggaa ttcactcctc aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc
     2641 caatgccctg gctcacaaat accactgaga tctttttccc tctgccaaaa attatgggga
     2701 catcatgaag ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc
     2761 aatagtgtgt tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca
     2821 tttaaaacat cagaatgagt atttggttta gagtttggca acatatgccc atatgctggc
     2881 tgccatgaac aaaggttggc tataaagagg tcatcagtat atgaaacagc cccctgctgt
     2941 ccattcctta ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt
     3001 tgtgttattt ttttctttaa catccctaaa attttcctta catgttttac tagccagatt
     3061 tttcctcctc tcctgactac tcccagtcat agctgtccct cttctcttat ggagatccct
     3121 cgacctgcag cccaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     3181 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     3241 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     3301 gaaacctgtc gtgccagcgg atccgcatct caattagtca gcaaccatag tcccgcccct
     3361 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     3421 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     3481 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctaactt gtttattgca
     3541 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     3601 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc
     3661 cgctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     3721 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     3781 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     3841 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     3901 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     3961 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4021 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4081 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4141 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4201 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4261 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     4321 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4381 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4441 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     4501 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4561 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     4621 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     4681 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     4741 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     4801 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     4861 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     4921 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     4981 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     5041 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     5101 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     5161 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     5221 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     5281 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     5341 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     5401 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     5461 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     5521 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     5581 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     5641 tgccacctgg
//
