LOCUS       sequence-177263-        5403 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For constructing NusA-SUMO-fusion proteins with N-terminal StrepII 
            tag and C-terminal His tag using ""blunt-end"" cloning..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5403)
  AUTHORS   Zhang Y, Fan Y
  TITLE     A Mutant Sumo Facilitates Quick Plasmid Construction for Expressing 
            Proteins with Native N-termini After Tag Removal.
  JOURNAL   Mol Biotechnol. 2017 May;59(4-5):159-167. doi: 
            10.1007/s12033-017-9998-6.
  PUBMED    28349302
REFERENCE   2  (bases 1 to 5403)
  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: "Mol 
            Biotechnol."; date: "2017-05"; volume: "59(4-5):159-167. doi"; 
            pages: " 10.1007/s12033-017-9998-6"
COMMENT     Sequence Label: pTNS2
FEATURES             Location/Qualifiers
     source          1..5403
                     /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..123
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             136..1617
                     /codon_start=1
                     /gene="nusA"
                     /product="transcription elongation protein (N utilization 
                     substance protein A)"
                     /label=NusA
                     /note="highly soluble in E. coli"
                     /translation="NKEILAVVEAVSNEKALPREKIFEALESALATATKKKYEQEIDVR
                     VQIDRKSGDFDTFRRWLVVDEVTQPTKEITLEAARYEDESLNLGDYVEDQIESVTFDRI
                     TTQTAKQVIVQKVREAERAMVVDQFREHEGEIITGVVKKVNRDNISLDLGNNAEAVILR
                     EDMLPRENFRPGDRVRGVLYSVRPEARGAQLFVTRSKPEMLIELFRIEVPEIGEEVIEI
                     KAAARDPGSRAKIAVKTNDKRIDPVGACVGMRGARVQAVSTELGGERIDIVLWDDNPAQ
                     FVINAMAPADVASIVVDEDKHTMDIAVEAGNLAQAIGRNGQNVRLASQLSGWELNVMTV
                     DDLQAKHQAEAHAAIDTFTKYLDIDEDFATVLVEEGFSTLEELAYVPMKELLEIEGLDE
                     PTVEALRERAKNALATIAQAQEESLGDNKPADDLLNLEGVDRDLAFKLAARGVCTLEDL
                     AEQGIDDLADIEGLTDEKAGALIMAARNICWFGDEA"
     CDS             1924..1941
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1957..2001
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2039..2057)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2053..2100
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2330..3145)
                     /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(2330..3145)
                     /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     3053..3072
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3146..3237)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3253..4002)
                     /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(3263..3280)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3503..3522)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4198..5280)
                     /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     5242..5261
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5281..5358)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctgta gaaataattt
       61 tgtttaactt taataaggag atataccatg agccacgtgt ggtcgcaccc tcaattcgag
      121 aaaggatcct ttaaaaacaa agaaattttg gctgtagttg aagccgtatc caatgaaaag
      181 gcgctacctc gcgagaagat tttcgaagca ttggaaagcg cgctggcgac agcaacaaag
      241 aaaaaatatg aacaagagat cgacgtccgc gtacagatcg atcgcaaaag cggtgatttt
      301 gacactttcc gtcgctggtt agttgttgat gaagtcaccc agccgaccaa ggaaatcacc
      361 cttgaagccg cacgttatga agatgaaagc ctgaacctgg gcgattacgt tgaagatcag
      421 attgagtctg ttacctttga ccgtatcact acccagacgg caaaacaggt tatcgtgcag
      481 aaagtgcgtg aagccgaacg tgcgatggtg gttgatcagt tccgtgaaca cgaaggtgaa
      541 atcatcaccg gcgtggtgaa aaaagtaaac cgcgacaaca tctctctgga tctgggcaac
      601 aacgctgaag ccgtgatcct gcgcgaagat atgctgccgc gtgaaaactt ccgccctggc
      661 gaccgcgttc gtggcgtgct ctattccgtt cgcccggaag cgcgtggcgc gcaactgttc
      721 gtcactcgtt ccaagccgga aatgctgatc gaactgttcc gtattgaagt gccagaaatc
      781 ggcgaagaag tgattgaaat taaagcagcg gctcgcgatc cgggttctcg tgcgaaaatc
      841 gcggtgaaaa ccaacgataa acgtatcgat ccggtaggtg cttgcgtagg tatgcgtggc
      901 gcgcgtgttc aggcggtgtc tactgaactg ggtggcgagc gtatcgatat cgtcctgtgg
      961 gatgataacc cggcgcagtt cgtgattaac gcaatggcac cggcagacgt tgcttctatc
     1021 gtggtggatg aagataaaca caccatggac atcgccgttg aagccggtaa tctggcgcag
     1081 gcgattggcc gtaacggtca gaacgtgcgt ctggcttcgc aactgagcgg ttgggaactc
     1141 aacgtgatga ccgttgacga cctgcaagct aagcatcagg cggaagcgca cgcagcgatc
     1201 gacaccttca ccaaatatct cgacatcgac gaagacttcg cgactgttct ggtagaagaa
     1261 ggcttctcga cgctggaaga attggcctat gtgccgatga aagagctgtt ggaaatcgaa
     1321 ggccttgatg agccgaccgt tgaagcactg cgcgagcgtg ctaaaaatgc actggccacc
     1381 attgcacagg cccaggaaga aagcctcggt gataacaaac cggctgacga tctgctgaac
     1441 cttgaagggg tagatcgtga tttggcattc aaactggccg cccgtggcgt ttgtacgctg
     1501 gaagatctcg ccgaacaggg cattgatgat ctggctgata tcgaagggtt gaccgacgaa
     1561 aaagccggag cactgattat ggctgcccgt aatatttgct ggttcggtga cgaagcgtac
     1621 gtaagatcct gggaagttaa accggaaacc cacatcaacc tgaaagtttc tgacggttct
     1681 tctgaaatct tcttcaaaat caaaaaaacc accccgctgc gtcgtctgat ggaagctttc
     1741 gctaaacgtc agggtaaaga aatggactct ctgcgtttcc tgtacgacgg tatccgtatc
     1801 caggctgacc agaccccgga agacctggac atggaagaca acgacatcat cgaagctcac
     1861 cgtgaacagc tgggtggtgc taccatggaa ttcgggcccg atatcggtac cctcgaggat
     1921 atccaccatc atcaccacca ttgagtcgag tctggtaaag aaaccgctgc tgcgaaattt
     1981 gaacgccagc acatggactc gtctactagc gcagcttaat taacctaggc tgctgccacc
     2041 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     2101 ctgaaacctc aggcatttga gaagcacacg gtcacactgc ttccggtagt caataaaccg
     2161 gtaaaccagc aatagacata agcggctatt taacgaccct gccctgaacc gacgacaagc
     2221 tgacgaccgg gtctccgcaa gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     2281 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact
     2341 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     2401 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     2461 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     2521 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     2581 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     2641 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     2701 gacgaaatac gcggtcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     2761 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     2821 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     2881 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     2941 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     3001 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     3061 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag
     3121 acgtttcccg ttgaatatgg ctcatactct tcctttttca atattattga agcatttatc
     3181 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     3241 gcatgcagcg ctcttccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc
     3301 ggcgagcggt gtcagctcac tcaaaagcgg taatacggtt atccacagaa tcaggggata
     3361 aagccggaaa gaacatgtga gcaaaaagca aagcaccgga agaagccaac gccgcaggcg
     3421 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagccagagg
     3481 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     3541 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     3601 agcgtggcgc tttctcatag ctcacgctgt tggtatctca gttcggtgta ggtcgttcgc
     3661 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     3721 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccatt
     3781 ggtaactgat ttagaggact ttgtcttgaa gttatgcacc tgttaaggct aaactgaaag
     3841 aacagatttt ggtgagtgcg gtcctccaac ccacttacct tggttcaaag agttggtagc
     3901 tcagcgaacc ttgagaaaac caccgttggt agcggtggtt tttctttatt tatgagatga
     3961 tgaatcaatc ggtctatcaa gtcaacgaac agctattccg ttactctaga tttcagtgca
     4021 atttatctct tcaaatgtag cacctgaagt cagccccata cgatataagt tgtaattctc
     4081 atgttagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     4141 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     4201 ctgcccgctt tccagtcggg aaacctgtcg tgccagttgc attaatgaat cggccaacgc
     4261 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     4321 gggcaacagt tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     4381 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     4441 tgagctgtct tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc
     4501 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     4561 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     4621 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     4681 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     4741 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     4801 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     4861 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     4921 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     4981 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     5041 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     5101 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     5161 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     5221 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     5281 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     5341 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     5401 aat
//
