LOCUS       sequence-111026-        5842 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  H6-TF-Smt3-GB1-NpuDnaEN102-CBD.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5842)
  AUTHORS   Guerrero F, Ciragan A, Iwai H
  TITLE     Tandem SUMO fusion vectors for improving soluble protein expression 
            and purification.
  JOURNAL   Protein Expr Purif. 2015 Aug 20. pii: S1046-5928(15)30039-5. doi: 
            10.1016/j.pep.2015.08.019.
  PUBMED    26297996
REFERENCE   2  (bases 1 to 5842)
  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: "Protein 
            Expr Purif. 2015 Aug 20. pii: S1046-5928(15)30039-5. doi: 
            10.1016/j.pep.2015.08.019."
COMMENT     Sequence Label: pCARSF85
FEATURES             Location/Qualifiers
     source          1..5842
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             121..1413
                     /codon_start=1
                     /product="ribosome-associated chaperone from E. coli 
                     (Hoffmann et al., 2010)"
                     /label=Trigger Factor
                     /translation="QVSVETTQGLGRRVTITIAADSIETAVKSELVNVAKKVRIDGFRK
                     GKVPMNIVAQRYGASVRQDVLGDLMSRNFIDAIIKEKINPAGAPTYVPGEYKLGEDFTY
                     SVEFEVYPEVELQGLEAIEVEKPIVEVTDADVDGMLDTLRKQQATWKEKDGAVEAEDRV
                     TIDFTGSVDGEEFEGGKASDFVLAMGQGRMIPGFEDGIKGHKAGEEFTIDVTFPEEYHA
                     ENLKGKAAKFAINLKKVEERELPELTAEFIKRFGVEDGSVEGLRAEVRKNMERELKSAI
                     RNRVKSQAIEGLVKANDIDVPAALIDSEIDVLRRQAAQRFGGNEKQALELPRELFEEQA
                     KRRVVVGLLLGEVIRTNELKADEERVKGLIEEMASAYEDPKEVIEFYSKNKELMDNMRN
                     VALEEQAVEAVLAKAKVTEKETTFNELMNQQA"
     CDS             1426..1716
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIETHREQIGG"
     CDS             2212..2367
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
                     PALWQLQ"
     CDS             2396..2440
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2478..2496)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2492..2539
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2769..3584)
                     /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(2769..3584)
                     /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     3492..3511
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3585..3676)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3692..4441)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(3702..3719)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3942..3961)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4637..5719)
                     /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     5681..5700
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5720..5797)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctgta gaaataattt
       61 tgtttaactt taataaggag atataccatg ggcagcagcc atcatcatca tcatcacggt
      121 caagtttcag ttgaaaccac tcaaggcctt ggccgccgtg taacgattac tatcgctgct
      181 gacagcatcg agaccgctgt taaaagcgag ctggtcaacg ttgcgaaaaa agtacgtatt
      241 gacggcttcc gcaaaggcaa agtgccaatg aatatcgttg ctcagcgtta tggcgcgtct
      301 gtacgccagg acgttctggg tgacctgatg agccgtaact tcattgacgc catcattaaa
      361 gaaaaaatca atccggctgg cgcaccgacc tatgttccgg gcgaatacaa gctgggtgaa
      421 gacttcacct actctgtaga gtttgaagtt tatccggaag ttgaactgca gggtctggaa
      481 gcgatcgaag ttgaaaaacc gatcgttgaa gtgaccgatg ctgacgttga cggcatgctg
      541 gatactctgc gtaaacagca ggcaacctgg aaagaaaaag acggcgctgt tgaagcagaa
      601 gaccgcgtaa ccatcgactt caccggttct gtagacggcg aagagttcga aggcggtaaa
      661 gcgtctgatt tcgtactggc gatgggccag ggtcgtatga tcccgggctt tgaagacggt
      721 atcaaaggcc acaaagctgg cgaagagttc accatcgacg tgaccttccc ggaagaatac
      781 cacgcagaaa acctgaaagg taaagcagcg aaattcgcta tcaacctgaa gaaagttgaa
      841 gagcgtgaac tgccggaact gactgcagaa ttcatcaaac gtttcggcgt tgaagatggt
      901 tccgtagaag gtctgcgcgc tgaagtgcgt aaaaacatgg agcgcgagct gaagagcgcc
      961 atccgtaacc gcgttaagtc tcaggcgatc gaaggtctgg taaaagctaa cgacatcgac
     1021 gtaccggctg cgctgatcga cagcgaaatc gacgttctgc gtcgccaggc tgcacagcgt
     1081 ttcggtggca acgaaaaaca agctctggaa ctgccgcgcg aactgttcga agaacaggct
     1141 aaacgtcgcg tagttgttgg cctgctgctg ggcgaagtta tccgcaccaa cgagctgaaa
     1201 gctgacgaag agcgcgtgaa aggcctgatc gaagagatgg cttctgcgta cgaagatccg
     1261 aaagaagtta tcgagttcta cagcaaaaac aaagaactga tggacaacat gcgcaatgtt
     1321 gctctggaag aacaggctgt tgaagctgta ctggcgaaag cgaaagtgac tgaaaaagaa
     1381 accactttca acgagctgat gaaccagcag gcgggtggag gtactagtga ctcagaagtc
     1441 aatcaagaag ctaagccaga ggtcaagcca gaagtcaagc ctgagactca catcaattta
     1501 aaggtgtccg atggatcttc agaaatcttc tttaagatca aaaagaccac tcctttaaga
     1561 aggctgatgg aagcgttcgc taaaagacag ggtaaggaaa tggactcctt aagattcttg
     1621 tacgacggta ttagaattca agctgatcag acccctgaag atttggacat ggaggataac
     1681 gatattattg agactcacag agaacagatt ggtggatccc atatgcagta caaacttatc
     1741 ctgaacggta aaaccctgaa aggtgaaacc accaccgaag ctgttgacgc tgctaccgcg
     1801 gaaaaagttt tcaaacagta cgctaacgac aacggtgttg acggtgaatg gacctacgac
     1861 gacgctacca aaacctacac cgtaacggaa ggatcctgtt taagctatga aacggaaata
     1921 ttgacagtag aatatggatt attaccgatt ggtaaaattg tagaaaagcg catcgaatgt
     1981 actgtttata gcgttgataa taatggaaat atttatacac aacctgtagc acaatggcac
     2041 gatcgcggag aacaagaggt gtttgagtat tgtttggaag atggttcatt gattcgggca
     2101 acaaaagacc ataagtttat gactgttgat ggtcaaatgt tgccaattga tgaaatattt
     2161 gaacgtgaat tggatttgat gcgggttgat aatttgccga acgaattcgg taccacaaat
     2221 cctggtgtat ccgcttggca ggtcaacaca gcttatactg cgggacaatt ggtcacatat
     2281 aacggcaaga cgtataaatg tttgcagccc cacacctcct tggcaggatg ggaaccatcc
     2341 aacgttcctg ccttgtggca gcttcaataa gcttgcggcc gcactcgagt ctggtaaaga
     2401 aaccgctgct gcgaaatttg aacgccagca catggactcg tctactagcg cagcttaatt
     2461 aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     2521 ggtcttgagg ggttttttgc tgaaacctca ggcatttgag aagcacacgg tcacactgct
     2581 tccggtagtc aataaaccgg taaaccagca atagacataa gcggctattt aacgaccctg
     2641 ccctgaaccg acgacaagct gacgaccggg tctccgcaag tggcactttt cggggaaatg
     2701 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     2761 attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     2821 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     2881 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     2941 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     3001 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     3061 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     3121 cgtgattgcg cctgagcgag acgaaatacg cggtcgctgt taaaaggaca attacaaaca
     3181 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     3241 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     3301 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     3361 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     3421 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     3481 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     3541 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcatactctt cctttttcaa
     3601 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     3661 tagaaaaata aacaaatagg catgcagcgc tcttccgctt cctcgctcac tgactcgcta
     3721 cgctcggtcg ttcgactgcg gcgagcggtg tcagctcact caaaagcggt aatacggtta
     3781 tccacagaat caggggataa agccggaaag aacatgtgag caaaaagcaa agcaccggaa
     3841 gaagccaacg ccgcaggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     3901 tcgacgctca agccagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     3961 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4021 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgtt ggtatctcag
     4081 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4141 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4201 gccactggca gcagccattg gtaactgatt tagaggactt tgtcttgaag ttatgcacct
     4261 gttaaggcta aactgaaaga acagattttg gtgagtgcgg tcctccaacc cacttacctt
     4321 ggttcaaaga gttggtagct cagcgaacct tgagaaaacc accgttggta gcggtggttt
     4381 ttctttattt atgagatgat gaatcaatcg gtctatcaag tcaacgaaca gctattccgt
     4441 tactctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc agccccatac
     4501 gatataagtt gtaattctca tgttagtcat gccccgcgcc caccggaagg agctgactgg
     4561 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     4621 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4681 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     4741 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     4801 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     4861 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatgt
     4921 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     4981 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     5041 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     5101 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     5161 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     5221 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     5281 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     5341 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     5401 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     5461 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     5521 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     5581 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     5641 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     5701 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     5761 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     5821 tgcgactcct gcattaggaa at
//
