LOCUS       sequence-358547-        5403 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression for NRBF2 MIT domain, residues 1-111. Has 
            N-terminal HIS-GST. Internal ID: WISP20-88..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5403)
  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 5403)
  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: pGEX-THTAP NRBF2 MIT residues 1-111
FEATURES             Location/Qualifiers
     source          1..5403
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(181..186,187..202,203..209)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    217..233
                     /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)."
     CDS             256..909
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     867..889
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             916..933
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             937..954
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             955..975
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             976..1020
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             1021..1044
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1451..1473)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1618..1636)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1704..1808
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1809..1877,1878..2669)
                     /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(2027..2046)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2840..3428
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3329..3348
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3582..3599
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3672..3749
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3750..4832
                     /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     complement(3769..3788)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4881..4886,4887..4904,4905..4911)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4919..4935
                     /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)."
     primer_bind     4943..4959
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4943..4959
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(4955..5403,1..79)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4975..4992)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4975..4991)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4984..5006)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 cttatcgact gcacggtgca ccaatgcttc tggcgtcagg cagccatcgg aagctgtggt
       61 atggctgtgc aggtcgtaaa tcactgcata attcgtgtcg ctcaaggcgc actcccgttc
      121 tggataatgt tttttgcgcc gacatcataa cggttctggc aaatattctg aaatgagctg
      181 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
      241 caggaaacag tattcatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
      301 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
      361 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
      421 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
      481 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
      541 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
      601 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
      661 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
      721 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
      781 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
      841 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
      901 cctccaaaat cggatctggt tccgcgtgga tctccgcacc atcaccatca ccatgaaaac
      961 ctgtattttc agggcggcct gaacgatatt tttgaagcgc agaaaattga atggcatgaa
     1021 cttgaagtcc tctttcaggg acccatggaa gtaatggaag gacccctcaa cctggctcat
     1081 caacagagca gacgagcaga ccgtttatta gctgcaggca aatacgaaga ggctatttct
     1141 tgtcacaaaa aggctgcagc atatctttct gaagccatga agctgacaca gtcagagcag
     1201 gctcatcttt cactggaatt gcaaagggat agccatatga aacagctcct cctcatccaa
     1261 gagagatgga aaagggccca gcgtgaagaa agattgaaag cccagcagaa cacagacaag
     1321 gatgcagctg cccatcttca gacatctcac aaaccctctg cagaggatgc agagggctga
     1381 caggatcctc gagaattcat cgtgactgac tgacgatctg cctcgcgcgt ttcggtgatg
     1441 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     1501 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg
     1561 cagccatgac ccagtcacgt agcgatagcg gagtgtataa ttcttgaaga cgaaagggcc
     1621 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     1681 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     1741 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     1801 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     1861 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     1921 tgggtgcacg agtaggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     1981 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     2041 tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     2101 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     2161 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     2221 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatggga gatcatgtaa
     2281 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     2341 ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     2401 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     2461 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     2521 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     2581 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     2641 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     2701 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     2761 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     2821 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     2881 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     2941 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     3001 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     3061 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     3121 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     3181 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     3241 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     3301 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     3361 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     3421 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     3481 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     3541 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     3601 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     3661 gcataaattc cgacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc
     3721 cggaagagag tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag
     3781 agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt
     3841 ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc
     3901 gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc
     3961 tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg
     4021 gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg
     4081 tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc
     4141 aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct
     4201 ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg
     4261 tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt
     4321 ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc
     4381 agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc
     4441 aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc
     4501 tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag
     4561 tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac
     4621 aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc
     4681 aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg
     4741 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     4801 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc
     4861 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
     4921 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgg attcactggc
     4981 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     5041 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     5101 ccaacagttg cgcagcctga atggcgaatg gcgctttgcc tggtttccgg caccagaagc
     5161 ggtgccggaa agctggctgg agtgcgatct tcctgaggcc gatactgtcg tcgtcccctc
     5221 aaactggcag atgcacggtt acgatgcgcc catctacacc aacgtgacct atcccattac
     5281 ggtcaatccg ccgtttgttc ccacggagaa tccgacgggt tgttactcgc tcacatttaa
     5341 tgttgatgaa agctggctac aggaaggcca gacgcgaatt atttttgatg gcgttggaat
     5401 tag
//
