LOCUS       sequence-403778-        7110 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Vps4 with stabilized MIT domain and 16aa GlyPro N-terminal 
            extension.
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  JOURNAL   Unpublished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            "Unpublished"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: 16_GP_Vps4p_L7C_A43C
FEATURES             Location/Qualifiers
     source          1..7110
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     368..387
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     621..638
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    653..792
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(718..737)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     878..900
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(894..1085)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2397..3479)
                     /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     3441..3460
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3480..3557)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3763..3782
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3866..3885
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3866..3884
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3885..3909
                     /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             3924..3946
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3957..3974
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3975..4016
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             4017..4037
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(5468..5486)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5482..5529
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(5767..5785)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5853..5957
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5958..6026,6027..6818)
                     /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(6176..6195)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6989..7110,1..467)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
       61 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
      121 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
      181 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
      241 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
      301 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
      361 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
      421 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
      481 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
      541 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
      601 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
      661 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac
      721 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
      781 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
      841 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
      901 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
      961 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     1021 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     1081 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     1141 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     1201 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     1261 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     1321 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     1381 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     1441 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     1501 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggccaggac
     1561 ccaacgctgc ccgagatgcg ccgcgtgcgg ctgctggaga tggcggacgc gatggatatg
     1621 ttctgccaag ggttggtttg cgcattcaca gttctccgca agaattgatt ggctccaatt
     1681 cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc cattcaggtc gaggtggccc
     1741 ggctccatgc accgcgacgc aacgcgggga ggcagacaag gtatagggcg gcgcctacaa
     1801 tccatgccaa cccgttccat gtgctcgccg aggcggcata aatcgccgtg acgatcagcg
     1861 gtccaatgat cgaagttagg ctggtaagag ccgcgagcga tccttgaagc tgtccctgat
     1921 ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc gggcatcccg atgccgccgg
     1981 aagcgagaag aatcataatg gggaaggcca tccagcctcg cgtcgcgaac gccagcaaga
     2041 cgtagcccag cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
     2101 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
     2161 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
     2221 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     2281 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     2341 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     2401 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     2461 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     2521 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     2581 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     2641 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
     2701 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     2761 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     2821 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     2881 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     2941 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     3001 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     3061 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     3121 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     3181 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     3241 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     3301 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     3361 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     3421 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     3481 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     3541 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     3601 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     3661 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     3721 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     3781 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     3841 gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat
     3901 aacaattccc ctctagaaat aattttgttt aactttaaga aggagatata catatgcatc
     3961 atcaccatca ccatggtaag cctatcccta accctctcct cggtctcgat tctacggaaa
     4021 acctgtattt tcagggaggc ggacctggag gcggtgggcc cggtggggga ggccctgggg
     4081 gtggcatgag cacgggagat ttttgtacta agggaataga actggttcaa aaggccattg
     4141 acctggacac agccacgcag tatgaggagg cgtatacagc atactataac ggattagatt
     4201 atttgatgtt gtgtttgaaa tacgagaaaa accctaagtc gaaggatttg ataagagcga
     4261 agtttacaga gtatctaaac cgtgcagagc agttgaaaaa gcacttggaa agtgaagagg
     4321 caaacgcggc aaagaaatct cctagcgctg gtagtggttc caacggtggt aataagaaaa
     4381 tctctcagga agagggtgag gataatggtg gagaagacaa caaaaagcta agaggtgcct
     4441 tatcgagtgc tattttgtct gagaagccca atgttaagtg ggaagacgtg gctggactgg
     4501 aaggagccaa agaggccctt aaagaagccg ttattttacc tgtcaagttc ccacatttgt
     4561 tcaaaggtaa ccgtaagccc acttcgggaa tcttattgta cggaccacca ggtacaggta
     4621 aatcatattt ggcaaaagca gtggccaccg aggccaattc tacctttttc agtgttagtt
     4681 ccagtgattt ggtttctaag tggatgggtg aatctgaaaa acttgtcaaa cagttatttg
     4741 cgatggcaag agaaaataaa ccttctatta tttttataga tgaagtggat gcgctaacag
     4801 gtaccagagg agaaggagag agcgaagcaa gtagaagaat taaaacagaa ttattggtac
     4861 aaatgaatgg tgttgggaat gactcccaag gtgttttggt ccttggcgca acaaatatcc
     4921 catggcaact ggacagtgcc atcaggagaa gatttgaaag aagaatatat ataccattac
     4981 cagatttggc ggcaagaacc accatgttcg agattaacgt cggcgacact ccatgcgtat
     5041 tgaccaagga agattataga actttaggtg caatgactga aggttattcc gggagtgata
     5101 ttgctgtagt ggtgaaagat gcgctaatgc aaccgataag aaaaattcaa agcgcgacac
     5161 atttcaaaga tgtctctacg gaggacgacg aaacaagaaa actaacacca tgctctccag
     5221 gtgatgatgg cgccatcgag atgagttgga cggatattga agctgatgaa ttaaaagaac
     5281 cagatctcac cataaaggat ttcttaaagg ctatcaaatc gacgagacct accgtgaatg
     5341 aggatgactt gctgaagcaa gaacagttca ctagagattt tggtcaagaa ggtaactagc
     5401 atatgaaggg cgagctcaga tccggctgct aacaaagccc gaaaggaagc tgagttggct
     5461 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     5521 ggttttttgc tgaaaggagg aactatatcc ggatatcccg caagaggccc ggcagtaccg
     5581 gcataaccaa gcctatgcct acagcatcca gggtgacggt gccgaggatg acgatgagcg
     5641 cattgttaga tttcatacac ggtgcctgac tgcgttagca atttaactgt gataaactac
     5701 cgcattaaag ctagcttatc gatgataagc tgtcaaacat gagaattaat tcttgaagac
     5761 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     5821 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     5881 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     5941 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     6001 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     6061 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     6121 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     6181 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact
     6241 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     6301 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     6361 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     6421 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     6481 agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     6541 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     6601 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     6661 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     6721 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     6781 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     6841 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     6901 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     6961 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     7021 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     7081 caactctttt tccgaaggta actggcttca
//
