LOCUS       sequence-111025-        4867 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  H6-TRX-Smt3-GB1-NpuDnaEN102-CBD.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4867)
  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 4867)
  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: pCARSF63
FEATURES             Location/Qualifiers
     source          1..4867
                     /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..444
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="SDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEI
                     ADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLD
                     ANLA"
     CDS             451..741
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIETHREQIGG"
     CDS             1237..1392
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
                     PALWQLQ"
     CDS             1421..1465
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1503..1521)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1517..1564
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1794..2609)
                     /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(1794..2609)
                     /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     2517..2536
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2610..2701)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2717..3466)
                     /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(2727..2744)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2967..2986)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3662..4744)
                     /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     4706..4725
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4745..4822)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctgta gaaataattt
       61 tgtttaactt taataaggag atataccatg ggcagcagcc atcatcatca tcatcacggc
      121 agcgataaaa ttattcacct gactgacgac agttttgaca cggatgtact caaagcggac
      181 ggggcgatcc tcgtcgattt ctgggcagag tggtgcggtc cgtgcaaaat gatcgccccg
      241 attctggatg aaatcgctga cgaatatcag ggcaaactga ccgttgcaaa actgaacatc
      301 gatcaaaacc ctggcactgc gccgaaatat ggcatccgtg gtatcccgac tctgctgctg
      361 ttcaaaaacg gtgaagtggc ggcaaccaaa gtgggtgcac tgtctaaagg tcagttgaaa
      421 gagttcctcg acgctaacct ggcgggaact agtgactcag aagtcaatca agaagctaag
      481 ccagaggtca agccagaagt caagcctgag actcacatca atttaaaggt gtccgatgga
      541 tcttcagaaa tcttctttaa gatcaaaaag accactcctt taagaaggct gatggaagcg
      601 ttcgctaaaa gacagggtaa ggaaatggac tccttaagat tcttgtacga cggtattaga
      661 attcaagctg atcagacccc tgaagatttg gacatggagg ataacgatat tattgagact
      721 cacagagaac agattggtgg atcccatatg cagtacaaac ttatcctgaa cggtaaaacc
      781 ctgaaaggtg aaaccaccac cgaagctgtt gacgctgcta ccgcggaaaa agttttcaaa
      841 cagtacgcta acgacaacgg tgttgacggt gaatggacct acgacgacgc taccaaaacc
      901 tacaccgtaa cggaaggatc ctgtttaagc tatgaaacgg aaatattgac agtagaatat
      961 ggattattac cgattggtaa aattgtagaa aagcgcatcg aatgtactgt ttatagcgtt
     1021 gataataatg gaaatattta tacacaacct gtagcacaat ggcacgatcg cggagaacaa
     1081 gaggtgtttg agtattgttt ggaagatggt tcattgattc gggcaacaaa agaccataag
     1141 tttatgactg ttgatggtca aatgttgcca attgatgaaa tatttgaacg tgaattggat
     1201 ttgatgcggg ttgataattt gccgaacgaa ttcggtacca caaatcctgg tgtatccgct
     1261 tggcaggtca acacagctta tactgcggga caattggtca catataacgg caagacgtat
     1321 aaatgtttgc agccccacac ctccttggca ggatgggaac catccaacgt tcctgccttg
     1381 tggcagcttc aataagcttg cggccgcact cgagtctggt aaagaaaccg ctgctgcgaa
     1441 atttgaacgc cagcacatgg actcgtctac tagcgcagct taattaacct aggctgctgc
     1501 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     1561 tttgctgaaa cctcaggcat ttgagaagca cacggtcaca ctgcttccgg tagtcaataa
     1621 accggtaaac cagcaataga cataagcggc tatttaacga ccctgccctg aaccgacgac
     1681 aagctgacga ccgggtctcc gcaagtggca cttttcgggg aaatgtgcgc ggaaccccta
     1741 tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa
     1801 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
     1861 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
     1921 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
     1981 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
     2041 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta
     2101 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
     2161 gcgagacgaa atacgcggtc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
     2221 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
     2281 aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga
     2341 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     2401 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
     2461 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg
     2521 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag
     2581 caagacgttt cccgttgaat atggctcata ctcttccttt ttcaatatta ttgaagcatt
     2641 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     2701 ataggcatgc agcgctcttc cgcttcctcg ctcactgact cgctacgctc ggtcgttcga
     2761 ctgcggcgag cggtgtcagc tcactcaaaa gcggtaatac ggttatccac agaatcaggg
     2821 gataaagccg gaaagaacat gtgagcaaaa agcaaagcac cggaagaagc caacgccgca
     2881 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagcca
     2941 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     3001 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     3061 gggaagcgtg gcgctttctc atagctcacg ctgttggtat ctcagttcgg tgtaggtcgt
     3121 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     3181 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     3241 cattggtaac tgatttagag gactttgtct tgaagttatg cacctgttaa ggctaaactg
     3301 aaagaacaga ttttggtgag tgcggtcctc caacccactt accttggttc aaagagttgg
     3361 tagctcagcg aaccttgaga aaaccaccgt tggtagcggt ggtttttctt tatttatgag
     3421 atgatgaatc aatcggtcta tcaagtcaac gaacagctat tccgttactc tagatttcag
     3481 tgcaatttat ctcttcaaat gtagcacctg aagtcagccc catacgatat aagttgtaat
     3541 tctcatgtta gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     3601 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     3661 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     3721 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     3781 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     3841 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     3901 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca
     3961 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     4021 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     4081 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     4141 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     4201 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     4261 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     4321 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     4381 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     4441 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     4501 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     4561 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     4621 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     4681 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     4741 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     4801 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     4861 aggaaat
//
