LOCUS       sequence-402888-        7062 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Vps4 with destabilized MIT domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7062)
  TITLE     Coupled mechanisms of Vps4 substrate recruitment and release from 
            autoinhibition
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7062)
  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            "Unpublished"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: Vps4p_L40D
FEATURES             Location/Qualifiers
     source          1..7062
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     154..171
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    186..325
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(251..270)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     411..433
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(427..618)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1930..3012)
                     /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     2974..2993
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3013..3090)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3296..3315
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3399..3418
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3399..3417
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3418..3442
                     /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             3457..3479
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3490..3507
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3508..3549
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3550..3570
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(4953..4971)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4967..5014
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(5252..5270)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5338..5442
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5443..5511,5512..6303)
                     /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(5661..5680)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6474..7062
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6963..6982
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
       61 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
      121 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
      181 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
      241 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
      301 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
      361 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
      421 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
      481 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
      541 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
      601 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
      661 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
      721 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
      781 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
      841 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
      901 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
      961 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     1021 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     1081 ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg cggctgctgg agatggcgga
     1141 cgcgatggat atgttctgcc aagggttggt ttgcgcattc acagttctcc gcaagaattg
     1201 attggctcca attcttggag tggtgaatcc gttagcgagg tgccgccggc ttccattcag
     1261 gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg ggaggcagac aaggtatagg
     1321 gcggcgccta caatccatgc caacccgttc catgtgctcg ccgaggcggc ataaatcgcc
     1381 gtgacgatca gcggtccaat gatcgaagtt aggctggtaa gagccgcgag cgatccttga
     1441 agctgtccct gatggtcgtc atctacctgc ctggacagca tggcctgcaa cgcgggcatc
     1501 ccgatgccgc cggaagcgag aagaatcata atggggaagg ccatccagcc tcgcgtcgcg
     1561 aacgccagca agacgtagcc cagcgcgtcg gccgccatgc cggcgataat ggcctgcttc
     1621 tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt
     1681 ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg
     1741 aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc
     1801 ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     1861 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     1921 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     1981 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     2041 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     2101 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     2161 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc
     2221 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     2281 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     2341 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     2401 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     2461 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     2521 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     2581 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     2641 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     2701 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     2761 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     2821 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     2881 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     2941 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     3001 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     3061 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
     3121 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     3181 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     3241 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     3301 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     3361 gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga
     3421 attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta agaaggagat
     3481 atacatatgc atcatcacca tcaccatggt aagcctatcc ctaaccctct cctcggtctc
     3541 gattctacgg aaaacctgta ttttcaggga atgagcacgg gagatttttt aactaaggga
     3601 atagaactgg ttcaaaaggc cattgacctg gacacagcca cgcagtatga ggaggcgtat
     3661 acagcatact ataacggatt agattatgat atgttggctt tgaaatacga gaaaaaccct
     3721 aagtcgaagg atttgataag agcgaagttt acagagtatc taaaccgtgc agagcagttg
     3781 aaaaagcact tggaaagtga agaggcaaac gcggcaaaga aatctcctag cgctggtagt
     3841 ggttccaacg gtggtaataa gaaaatctct caggaagagg gtgaggataa tggtggagaa
     3901 gacaacaaaa agctaagagg tgccttatcg agtgctattt tgtctgagaa gcccaatgtt
     3961 aagtgggaag acgtggctgg actggaagga gccaaagagg cccttaaaga agccgttatt
     4021 ttacctgtca agttcccaca tttgttcaaa ggtaaccgta agcccacttc gggaatctta
     4081 ttgtacggac caccaggtac aggtaaatca tatttggcaa aagcagtggc caccgaggcc
     4141 aattctacct ttttcagtgt tagttccagt gatttggttt ctaagtggat gggtgaatct
     4201 gaaaaacttg tcaaacagtt atttgcgatg gcaagagaaa ataaaccttc tattattttt
     4261 atagatgaag tggatgcgct aacaggtacc agaggagaag gagagagcga agcaagtaga
     4321 agaattaaaa cagaattatt ggtacaaatg aatggtgttg ggaatgactc ccaaggtgtt
     4381 ttggtccttg gcgcaacaaa tatcccatgg caactggaca gtgccatcag gagaagattt
     4441 gaaagaagaa tatatatacc attaccagat ttggcggcaa gaaccaccat gttcgagatt
     4501 aacgtcggcg acactccatg cgtattgacc aaggaagatt atagaacttt aggtgcaatg
     4561 actgaaggtt attccgggag tgatattgct gtagtggtga aagatgcgct aatgcaaccg
     4621 ataagaaaaa ttcaaagcgc gacacatttc aaagatgtct ctacggagga cgacgaaaca
     4681 agaaaactaa caccatgctc tccaggtgat gatggcgcca tcgagatgag ttggacggat
     4741 attgaagctg atgaattaaa agaaccagat ctcaccataa aggatttctt aaaggctatc
     4801 aaatcgacga gacctaccgt gaatgaggat gacttgctga agcaagaaca gttcactaga
     4861 gattttggtc aagaaggtaa ctagcatatg aagggcgagc tcagatccgg ctgctaacaa
     4921 agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag cataacccct
     4981 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggata
     5041 tcccgcaaga ggcccggcag taccggcata accaagccta tgcctacagc atccagggtg
     5101 acggtgccga ggatgacgat gagcgcattg ttagatttca tacacggtgc ctgactgcgt
     5161 tagcaattta actgtgataa actaccgcat taaagctagc ttatcgatga taagctgtca
     5221 aacatgagaa ttaattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt
     5281 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     5341 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     5401 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     5461 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     5521 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     5581 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     5641 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa
     5701 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     5761 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     5821 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     5881 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     5941 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca
     6001 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     6061 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     6121 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     6181 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     6241 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     6301 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     6361 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     6421 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     6481 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     6541 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     6601 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     6661 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     6721 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     6781 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     6841 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     6901 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     6961 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     7021 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aa
//
