LOCUS       sequence-402891-        7110 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Vps4 with stabilized MIT domain with an N-terminal extension of 16 
            binding amino acids.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7110)
  TITLE     Coupled mechanisms of Vps4 substrate recruitment and release from 
            autoinhibition
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7110)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: 2xPeptide 8_Vps4p_L7C_A43C
FEATURES             Location/Qualifiers
     source          1..7110
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    775..914
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(840..859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1000..1022
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1016..1207)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2519..3601)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     3563..3582
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3602..3679)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3885..3904
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3988..4007
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3988..4006
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4007..4031
                     /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)."
     RBS             4046..4068
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4079..4096
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4097..4138
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             4139..4159
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(5590..5608)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5604..5651
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(5889..5907)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5975..6079
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6080..6148,6149..6940)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6298..6317)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
      841 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
      901 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
      961 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     1021 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     1081 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     1141 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     1201 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     1261 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     1321 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     1381 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     1441 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     1501 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     1561 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     1621 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg
     1681 acccaacgct gcccgagatg cgccgcgtgc ggctgctgga gatggcggac gcgatggata
     1741 tgttctgcca agggttggtt tgcgcattca cagttctccg caagaattga ttggctccaa
     1801 ttcttggagt ggtgaatccg ttagcgaggt gccgccggct tccattcagg tcgaggtggc
     1861 ccggctccat gcaccgcgac gcaacgcggg gaggcagaca aggtataggg cggcgcctac
     1921 aatccatgcc aacccgttcc atgtgctcgc cgaggcggca taaatcgccg tgacgatcag
     1981 cggtccaatg atcgaagtta ggctggtaag agccgcgagc gatccttgaa gctgtccctg
     2041 atggtcgtca tctacctgcc tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc
     2101 ggaagcgaga agaatcataa tggggaaggc catccagcct cgcgtcgcga acgccagcaa
     2161 gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     2221 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     2281 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     2341 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     2401 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     2461 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     2521 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     2581 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     2641 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     2701 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     2761 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc
     2821 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     2881 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     2941 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     3001 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     3061 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     3121 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     3181 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     3241 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     3301 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     3361 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     3421 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     3481 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     3541 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     3601 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     3661 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     3721 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     3781 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     3841 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     3901 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     3961 aggatcgaga tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg
     4021 ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata tacatatgca
     4081 tcatcaccat caccatggta agcctatccc taaccctctc ctcggtctcg attctacgga
     4141 aaacctgtat tttcagggac ttgttaaaaa tgttattgaa gatcttgtta aaaatgttat
     4201 tgaagatatg agcacgggag atttttgtac taagggaata gaactggttc aaaaggccat
     4261 tgacctggac acagccacgc agtatgagga ggcgtataca gcatactata acggattaga
     4321 ttatttgatg ttgtgtttga aatacgagaa aaaccctaag tcgaaggatt tgataagagc
     4381 gaagtttaca gagtatctaa accgtgcaga gcagttgaaa aagcacttgg aaagtgaaga
     4441 ggcaaacgcg gcaaagaaat ctcctagcgc tggtagtggt tccaacggtg gtaataagaa
     4501 aatctctcag gaagagggtg aggataatgg tggagaagac aacaaaaagc taagaggtgc
     4561 cttatcgagt gctattttgt ctgagaagcc caatgttaag tgggaagacg tggctggact
     4621 ggaaggagcc aaagaggccc ttaaagaagc cgttatttta cctgtcaagt tcccacattt
     4681 gttcaaaggt aaccgtaagc ccacttcggg aatcttattg tacggaccac caggtacagg
     4741 taaatcatat ttggcaaaag cagtggccac cgaggccaat tctacctttt tcagtgttag
     4801 ttccagtgat ttggtttcta agtggatggg tgaatctgaa aaacttgtca aacagttatt
     4861 tgcgatggca agagaaaata aaccttctat tatttttata gatgaagtgg atgcgctaac
     4921 aggtaccaga ggagaaggag agagcgaagc aagtagaaga attaaaacag aattattggt
     4981 acaaatgaat ggtgttggga atgactccca aggtgttttg gtccttggcg caacaaatat
     5041 cccatggcaa ctggacagtg ccatcaggag aagatttgaa agaagaatat atataccatt
     5101 accagatttg gcggcaagaa ccaccatgtt cgagattaac gtcggcgaca ctccatgcgt
     5161 attgaccaag gaagattata gaactttagg tgcaatgact gaaggttatt ccgggagtga
     5221 tattgctgta gtggtgaaag atgcgctaat gcaaccgata agaaaaattc aaagcgcgac
     5281 acatttcaaa gatgtctcta cggaggacga cgaaacaaga aaactaacac catgctctcc
     5341 aggtgatgat ggcgccatcg agatgagttg gacggatatt gaagctgatg aattaaaaga
     5401 accagatctc accataaagg atttcttaaa ggctatcaaa tcgacgagac ctaccgtgaa
     5461 tgaggatgac ttgctgaagc aagaacagtt cactagagat tttggtcaag aaggtaacta
     5521 gcatatgaag ggcgagctca gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     5581 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     5641 ggggtttttt gctgaaagga ggaactatat ccggatatcc cgcaagaggc ccggcagtac
     5701 cggcataacc aagcctatgc ctacagcatc cagggtgacg gtgccgagga tgacgatgag
     5761 cgcattgtta gatttcatac acggtgcctg actgcgttag caatttaact gtgataaact
     5821 accgcattaa agctagctta tcgatgataa gctgtcaaac atgagaatta attcttgaag
     5881 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     5941 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     6001 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     6061 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     6121 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     6181 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     6241 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     6301 atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca
     6361 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     6421 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     6481 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     6541 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     6601 cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg
     6661 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     6721 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     6781 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     6841 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     6901 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     6961 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     7021 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     7081 agaccccgta gaaaagatca aaggatcttc
//
