LOCUS       sequence-460673-        5937 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His6-SUMO^NIT-FraB where ""NIT"" stands for ""no internal 
            translation"", reflecting the mutation of the two 
            Shine-Dalgarno-like sequences within the SUMO tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5937)
  TITLE     Design of a yeast SUMO tag to eliminate internal translation 
            initiation
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5937)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pLANT-2b
FEATURES             Location/Qualifiers
     source          1..5937
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      319..864
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     765..784
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     981..998
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     2649..2668
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2752..2771
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2752..2770
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2771..2795
                     /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             2810..2832
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2843..2860
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2861..3154
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDLQNNDIIEAHREQIAG"
     terminator      4145..4192
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     tRNA            4429..4505
                     /gene="argU"
                     /product="E. coli tRNA-Arg recognizing AGA and AGG"
                     /label=argU
     CDS             complement(4959..5774)
                     /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(4959..5774)
                     /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     5682..5701
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5766..5785
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca cgatgataag ctgtcaaaca tgagaattac aacttatatc gtatggggct
      241 gacttcaggt gctacatttg aagagataaa ttgcactgaa atctagaaat attttatctg
      301 attaataaga tgatcttctt gagatcgttt tggtctgcgc gtaatctctt gctctgaaaa
      361 cgaaaaaacc gccttgcagg gcggtttttc gaaggttctc tgagctacca actctttgaa
      421 ccgaggtaac tggcttggag gagcgcagtc accaaaactt gtcctttcag tttagcctta
      481 accggcgcat gacttcaaga ctaactcctc taaatcaatt accagtggct gctgccagtg
      541 gtgcttttgc atgtctttcc gggttggact caagacgata gttaccggat aaggcgcagc
      601 ggtcggactg aacggggggt tcgtgcatac agtccagctt ggagcgaact gcctacccgg
      661 aactgagtgt caggcgtgga atgagacaaa cgcggccata acagcggaat gacaccggta
      721 aaccgaaagg caggaacagg agagcgcacg agggagccgc cagggggaaa cgcctggtat
      781 ctttatagtc ctgtcgggtt tcgccaccac tgatttgagc gtcagatttc gtgatgcttg
      841 tcaggggggc ggagcctatg gaaaaacggc tttgccgcgg ccctctcact tccctgttaa
      901 gtatcttcct ggcatcttcc aggaaatctc cgccccgttc gtaagccatt tccgctcgcc
      961 gcagtcgaac gaccgagcgt agcgagtcag tgagcgagga agcggaatat atcctgtatc
     1021 acatattctg ctgacgcacc ggtgcagcct tttttctcct gccacatgaa gcgattcaca
     1081 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     1141 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     1201 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     1261 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     1321 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     1381 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     1441 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
     1501 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
     1561 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
     1621 ggtcctcaac gacaggagca cgatcatgcg cacccgtggc caggacccaa cgctgcccga
     1681 gatgcgccgc gtgcggctgc tggagatggc ggacgcgatg gatatgttct gccaagggtt
     1741 ggtttgcgca ttcacagttc tccgcaagaa ttgattggct ccaattcttg gagtggtgaa
     1801 tccgttagcg aggtgccgcc ggcttccatt caggtcgagg tggcccggct ccatgcaccg
     1861 cgacgcaacg cggggaggca gacaaggtat agggcggcgc ctacaatcca tgccaacccg
     1921 ttccatgtgc tcgccgaggc ggcataaatc gccgtgacga tcagcggtcc aatgatcgaa
     1981 gttaggctgg taagagccgc gagcgatcct tgaagctgtc cctgatggtc gtcatctacc
     2041 tgcctggaca gcatggcctg caacgcgggc atcccgatgc cgccggaagc gagaagaatc
     2101 ataatgggga aggccatcca gcctcgcgtc gcgaacgcca gcaagacgta gcccagcgcg
     2161 tcggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca
     2221 gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccacaagcga caggccgatc
     2281 atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc
     2341 tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc
     2401 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgacgctct
     2461 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     2521 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     2581 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     2641 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
     2701 ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac
     2761 tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact
     2821 ttaagaagga gatatacata tgcatcatca tcatcatcat atgtcggact cagaagtcaa
     2881 tcaagaagct aagccagagg tcaagccaga agtcaagcct gagactcaca tcaatttaaa
     2941 ggtgtccgat ggatcttcag agatcttctt caagatcaaa aagaccactc ctttaagaag
     3001 gctgatggaa gcgttcgcta aaagacaggg taaggaaatg gactccttaa gattcttgta
     3061 cgacggtatt agaatccaag ctgatcagac ccctgaagat ttggacctcc agaacaacga
     3121 tattattgag gctcacagag aacagattgc tggcatgatg ggtatgaaag agacagttag
     3181 caatattgtg accagccagg cagagaaagg aggcgttaaa cacgtctatt acgtggcgtg
     3241 cggcggttct tatgcggcgt tctatccggc gaaagcattt ttagaaaaag aagcgaaagc
     3301 gttgactgtc ggtctgtata acagcggaga atttattaac aacccgccgg tagcgctggg
     3361 agaaaatgcc gttgtggttg tcgcctccca caaaggtaat acgccagaga caattaaagc
     3421 ggctgaaatc gcccgtcagc acggcgcgcc ggtcattggt ttaacctgga taatggattc
     3481 accgttggtg gcgcattgcg actatgtgga aacgtacacg tttggcgacg gtaaagatat
     3541 tgccggagag aaaacgatga aaggcctgct gagtgcggtc gaactgctcc agcagacgga
     3601 agggtatgcg cactacgacg attttcagga tggcgtcagc aaaatcaacc gtatcgtctg
     3661 gcgcgcttgc gagcaggtag cggagcgtgc gcaggcgttc gcgcaggaat ataaagacga
     3721 taaagtcatt tataccgtcg ccagcggcgc gggctatggc gcagcctacc tacagagcat
     3781 ctgcatcttt atggaaatgc aatggataca ttccgcctgt attcatagcg gtgagttttt
     3841 ccacgggccg tttgaaatta ccgatgcgaa tacgcctttc ttcttccagt tttccgaggg
     3901 caatacgcgg gcggtggatg aacgcgcgtt aaacttcctg aaaaaatatg gccgccggat
     3961 tgaagttgtc gatgcgaaag aactggggct atcgaccatt aaaaccacgg ttattgatta
     4021 ctttaaccac tctctcttta ataacgttta tcccgtttac aatcgggcgt tagctgaggc
     4081 gcgtcagcat ccgttaacga cgcgccgcta tatgtggaaa gtggaatatt aagctgagca
     4141 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     4201 aggaactata tccggatatc ccgcaagagg cccggcagta ccggcataac caagcctatg
     4261 cctacagcat ccagggtgac ggtgctgtac aattgacgac gacaagggcc cgggcaaact
     4321 agtaatcaga cgcggtcgtt cacttgttca gcaaccagat caaaagccat tgactcagca
     4381 agggttgacc gtataattca cgcgattaca ccgcattgcg gtatcaacgc gcccttagct
     4441 cagttggata gagcaacgac cttctaagtc gtgggccgca ggttcgaatc ctgcagggcg
     4501 cgccattaca attcaatcag ttacgccttc tttatatcct ccagccatgg ccttgaaatg
     4561 gcgttagtca tgaaatatag accgccatcg agtacccctt gtacccttaa ctcttcctga
     4621 tacgtaaata atgatttggt ggcccttgct ggacttgaac cagcgaccaa gcgattatga
     4681 gtcgcctgct ctaaccactg agctaaaggg ccttgagtgt gcaataacaa tacttataaa
     4741 ccacgcaata aacatgatga tctagagaat cccgtcgtag ccaccatctt tttttgcggg
     4801 agtggcgaaa ttggtagacg caccagattt aggttctggc gccgctaggt gtgcgagttc
     4861 aagtctcgcc tcccgcacca ttcaccagaa agcgttgatc ggatgccctc gagtcgggca
     4921 gcgttgggtc ctggccacgg gtgcgcatga tcgaattctt agaaaaactc atcgagcatc
     4981 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
     5041 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
     5101 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
     5161 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
     5221 agcttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
     5281 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg
     5341 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
     5401 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
     5461 gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
     5521 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
     5581 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
     5641 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
     5701 ccatataaat cagcatccat gttggaattt aatcgcggcc tcgagcaaga cgtttcccgt
     5761 tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt
     5821 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     5881 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaac
//
