LOCUS       sequence-358645-        5828 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression for KATNA1 MIT domains, residues 1-79. Has 
            N-terminal HIS-SUMO tag. Internal ID: WISP19-20..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5828)
  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 5828)
  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: pCA528 KATNA1 MIT residues 1-79
FEATURES             Location/Qualifiers
     source          1..5828
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     65..84
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     168..187
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        168..186
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    187..211
                     /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             226..248
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             262..279
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             283..573
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             860..877
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(930..948)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      944..991
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1028..1483
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1115..1134)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1325..1346
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1575..2390)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFKVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2298..2317
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2382..2401
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2512..3100
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3001..3020
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3254..3271
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3286..3425
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3351..3370)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3511..3533
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3527..3718)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4527..5609)
                     /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
                     YIPPLTTIKQDFRLLGQTSVDSLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5571..5590
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5610..5687)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
       61 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
      121 ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac
      181 tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa
      241 gaaggagata tacatatggg tcatcaccat catcaccatg gttcggactc agaagtcaat
      301 caagaagcta agccagaggt caagccagaa gtcaagcctg agactcacat caatttaaag
      361 gtgtccgatg gatcttcaga gatcttcttc aagatcaaaa agaccactcc tttaagaagg
      421 ctgatggaag cgttcgctaa aagacagggt aaggaaatgg actccttaag attcttgtac
      481 gacggtatta gaattcaagc tgatcagacc cctgaagatt tggacatgga ggataacgat
      541 attattgagg ctcacagaga acagattggt gggatgagtc ttcttatgat tagtgagaat
      601 gtaaaattgg ctcgtgaata tgcattgctg ggaaactatg actctgcgat ggtctattat
      661 cagggagttc ttgaccaaat gaacaagtat ctgtactcag tcaaagatac atacctccag
      721 cagaaatggc aacaggtttg gcaggaaata aatgtggaag ctaaacatgt taaagatatc
      781 atgaaaacac tagagagctt taaactggac tgaggatccg aattcgagct ccgtcgacaa
      841 gcttgcggcc gcactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc
      901 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
      961 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc
     1021 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     1081 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     1141 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     1201 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     1261 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     1321 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     1381 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     1441 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     1501 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     1561 tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata
     1621 tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca
     1681 ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca
     1741 acatcaatac aacctattaa ttttccctcg tcaaaaataa ggttatcaag tgagaaatca
     1801 ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact
     1861 tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta
     1921 ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta
     1981 caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca
     2041 cctgaatcag gatattcttc taataccttg aatgctgttt tcccggggat cgcagtggtg
     2101 agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat
     2161 tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg
     2221 ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca
     2281 cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg
     2341 gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt
     2401 gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga
     2461 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     2521 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     2581 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     2641 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     2701 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     2761 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     2821 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     2881 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     2941 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     3001 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     3061 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     3121 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     3181 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     3241 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     3301 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
     3361 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     3421 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     3481 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     3541 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     3601 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     3661 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     3721 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     3781 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     3841 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     3901 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     3961 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     4021 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     4081 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     4141 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg
     4201 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     4261 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     4321 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     4381 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     4441 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     4501 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4561 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     4621 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     4681 ccctgagaga gttgcagcaa gctgtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     4741 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     4801 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     4861 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     4921 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     4981 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     5041 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     5101 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     5161 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     5221 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     5281 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     5341 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     5401 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     5461 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     5521 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     5581 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     5641 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     5701 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     5761 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     5821 ggggcctg
//
