LOCUS       sequence-349445-        6523 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of His6-SUMO-TEV-GFP-PreScission-gamma-TuNA in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6523)
  AUTHORS   Wieczorek M, Urnavicius L, Ti SC, Molloy KR, Chait BT, Kapoor TM
  TITLE     Asymmetric Molecular Architecture of the Human gamma-Tubulin Ring 
            Complex.
  JOURNAL   Cell. 2020 Jan 9;180(1):165-175.e16. doi: 
            10.1016/j.cell.2019.12.007. Epub 2019 Dec 17.
  PUBMED    31862189
REFERENCE   2  (bases 1 to 6523)
  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"; doi: 
            "10.1016/j.cell.2019.12.007"; journalName: "Cell"; date: "2020-01-9-
            9"; volume: "180"; issue: "1"; pages: "165-175.e16"
COMMENT     Sequence Label: pMW96
FEATURES             Location/Qualifiers
     source          1..6523
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(140..159)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     487..508
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             566..589
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             758..775
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(828..846)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      842..889
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      926..1381
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1013..1032)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1223..1244
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1474..2289)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1474..2289)
                     /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
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2197..2216
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2281..2300
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2411..2999
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2900..2919
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3153..3170
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3185..3324
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3250..3269)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3410..3432
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3426..3617)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4426..5508)
                     /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     5470..5489
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5509..5586)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5792..5811
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5895..5914
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5895..5913
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5914..5938
                     /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             5953..5975
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5994..6011
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6030..6323
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             6330..6350
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(6357..6359,6360..6362,6363..6523,1..550)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(6357..6359,6360..6362,6363..6523,1..550)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6402..6423)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
ORIGIN
        1 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
       61 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
      121 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
      181 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
      241 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
      301 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
      361 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
      421 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
      481 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
      541 gctgtacaag gtcgacggtt cttctctgga agttctgttc caggggccca cagtgtctcc
      601 caccagagca cggaacatga aggactttga aaatcaaatc actgaattga agaaagaaaa
      661 ctttaaccta aagctccgca tctatttcct tgaggaaaga atgcaacagg aatttcatgg
      721 ccccactgaa catatctact gagcggccgc actcgagcac caccaccacc accactgaga
      781 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata
      841 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
      901 aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
      961 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     1021 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     1081 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     1141 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     1201 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     1261 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     1321 aatgagctga tttaacaaaa atttaacgcg aattttaaca aactagtaac gtttacaatt
     1381 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     1441 cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg
     1501 aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg
     1561 taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc
     1621 tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag
     1681 gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt
     1741 atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact
     1801 cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc
     1861 gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag
     1921 cgcatcaaca atgttttcac ctgaatcagg atattcttct aatacctgga atgctgtttt
     1981 cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat
     2041 ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc
     2101 attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata
     2161 caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata
     2221 taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat
     2281 atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga
     2341 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     2401 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     2461 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2521 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     2581 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     2641 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     2701 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     2761 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     2821 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     2881 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     2941 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     3001 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     3061 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     3121 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     3181 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
     3241 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     3301 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     3361 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     3421 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     3481 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     3541 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     3601 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     3661 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     3721 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     3781 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     3841 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     3901 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     3961 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     4021 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     4081 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
     4141 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     4201 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
     4261 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     4321 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     4381 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     4441 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4501 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     4561 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     4621 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     4681 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     4741 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     4801 accctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     4861 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     4921 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     4981 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     5041 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     5101 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     5161 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     5221 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     5281 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     5341 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     5401 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     5461 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     5521 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     5581 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     5641 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     5701 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     5761 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     5821 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     5881 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     5941 tctagaaata attttgttta actttaagaa ggagatatac catgggttct tctcaccatc
     6001 accatcacca tggttcttct atggctagca tgtcggactc agaagtcaat caagaagcta
     6061 agccagaggt caagccagaa gtcaagcctg agactcacat caatttaaag gtgtccgatg
     6121 gatcttcaga gatcttcttc aagatcaaaa agaccactcc tttaagaagg ctgatggaag
     6181 cgttcgctaa aagacagggt aaggaaatgg actccttaag attcttgtac gacggtatta
     6241 gaattcaagc tgatcagacc cctgaagatt tggacatgga ggataacgat attattgagg
     6301 ctcacagaga acagattggt gggatcgagg aaaacctgta cttccaatcc aatattatgg
     6361 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     6421 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     6481 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgt
//
