LOCUS       sequence-339141-        7715 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of soluble E. coli Tus protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7715)
  AUTHORS   Norouzi M, Panfilov S, Pardee K
  TITLE     High-Efficiency Protection of Linear DNA in Cell-Free Extracts from 
            Escherichia coli and Vibrio natriegens.
  JOURNAL   ACS Synth Biol. 2021 Jun 23. doi: 10.1021/acssynbio.1c00110.
  PUBMED    34161082
REFERENCE   2  (bases 1 to 7715)
  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: "ACS Synth 
            Biol. 2021 Jun 23. doi: 10.1021/acssynbio.1c00110."
COMMENT     Sequence Label: pET24_NusA-Tus
FEATURES             Location/Qualifiers
     source          1..7715
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     47..66
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        47..65
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    66..90
                     /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             104..126
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             137..154
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             155..1636
                     /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             1658..1678
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(2696..2714)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2710..2757
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2794..3249
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2881..2900)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3091..3112
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3341..4156)
                     /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(3341..4156)
                     /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     4064..4083
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4148..4167
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4278..4866
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4767..4786
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5020..5037
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5052..5191
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5117..5136)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5277..5299
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5293..5484)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6293..7375)
                     /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     7337..7356
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7376..7453)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7659..7678
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact
       61 ataggggaat tgtgagcgga taacaattcc cctctagaat aattttgttt aactttaaga
      121 aggagatata catatgcatc accatcacca tcacaacaaa gaaattttgg ctgtagttga
      181 agccgtatcc aatgaaaagg cgctacctcg cgagaagatt ttcgaagcat tggaaagcgc
      241 gctggcgaca gcaacaaaga aaaaatatga acaagagatc gacgtccgcg tacagatcga
      301 tcgcaaaagc ggtgattttg acactttccg tcgctggtta gttgttgatg aagtcaccca
      361 gccgaccaag gaaatcaccc ttgaagccgc acgttatgaa gatgaaagcc tgaacctggg
      421 cgattacgtt gaagatcaga ttgagtctgt tacctttgac cgtatcacta cccagacggc
      481 aaaacaggtt atcgtgcaga aagtgcgtga agccgaacgt gcgatggtgg ttgatcagtt
      541 ccgtgaacac gaaggtgaaa tcatcaccgg cgtggtgaaa aaagtaaacc gcgacaacat
      601 ctctctggat ctgggcaaca acgctgaagc cgtgatcctg cgcgaagata tgctgccgcg
      661 tgaaaacttc cgccctggcg accgcgttcg tggcgtgctc tattccgttc gcccggaagc
      721 gcgtggcgcg caactgttcg tcactcgttc caagccggaa atgctgatcg aactgttccg
      781 tattgaagtg ccagaaatcg gcgaagaagt gattgaaatt aaagcagcgg ctcgcgatcc
      841 gggttctcgt gcgaaaatcg cggtgaaaac caacgataaa cgtatcgatc cggtaggtgc
      901 ttgcgtaggt atgcgtggcg cgcgtgttca ggcggtgtct actgaactgg gtggcgagcg
      961 tatcgatatc gtcctgtggg atgataaccc ggcgcagttc gtgattaacg caatggcacc
     1021 ggcagacgtt gcttctatcg tggtggatga agataaacac accatggata tcgccgttga
     1081 agccggtaac ctggcgcagg cgattggccg taacggtcag aacgtgcgtc tggcttcgca
     1141 gctgagcggt tgggaactca acgtgatgac cgttgacgac ctgcaggcta agcatcaggc
     1201 ggaagcgcac gcagcgatcg acaccttcac caaatatctc gacatcgacg aagacttcgc
     1261 gactgttctg gtagaagaag gcttctcgac gctggaagaa ttggcctatg tgccgatgaa
     1321 agagctgttg gaaatcgaag gccttgatga gccgaccgtt gaagcactgc gcgagcgtgc
     1381 taaaaatgca ctggccacca ttgcacaggc ccaggaagaa agcctcggtg ataacaaacc
     1441 ggctgacgat ctgctgaacc ttgaaggggt agatcgtgat ttggcattca aactggccgc
     1501 ccgtggcgtt tgtacgctgg aagatctcgc cgaacagggc attgatgatc tggctgatat
     1561 cgaagggttg accgacgaaa aagccggagc actgattatg gctgcccgta acatttgctg
     1621 gttcggtgac gaagcggatt acgacatccc aacgaccgaa aacctgtatt ttcagggcat
     1681 ggcgcgttac gatctcgtag accgactcaa cactaccttt cgccagatgg aacaagagct
     1741 ggctatattt gccgctcatc ttgagcaaca caagctattg gttgcccgcg tgttctcttt
     1801 gccggaggta aaaaaagagg atgagcataa tccgcttaat cgtattgagg taaaacaaca
     1861 tctcggcaac gacgcgcagt cgctggcgtt gcgtcatttc cgccatttat ttattcaaca
     1921 acagtccgaa aatcgcagca gcaaggccgc tgtccgtctg cctggcgtgt tgtgttacca
     1981 ggtcgataac ctttcgcaag cagcgttggt cagtcatatt cagcacatca ataaactcaa
     2041 gaccacgttc gagcatatcg tcacggttga atcagaactc cccaccgcgg cacgttttga
     2101 atgggtgcat cgtcatttgc cggggctgat cacccttaat gcttaccgca cgctcaccgt
     2161 tctgcacgac cccgccactt tacgctttgg ttgggctaat aaacatatca ttaagaattt
     2221 acatcgtgat gaagtcctgg cacagctgga aaaaagcctg aaatcaccac gcagtgtcgc
     2281 accgtggacg cgcgaggagt ggcaaagaaa actggagcga gagtatcagg atatcgctgc
     2341 cctgccacag aacgcgaagt taaaaatcaa acgtccggtg aaggtgcagc cgattgcccg
     2401 cgtctggtac aaaggagatc aaaaacaagt ccaacacgcc tgccctacac cactgattgc
     2461 actgattaat cgggataatg gcgcgggcgt gccggacgtt ggtgagttgt taaattacga
     2521 tgccgacaat gtgcagcacc gttataaacc tcaggcgcag ccgcttcgtt tgatcattcc
     2581 acggctgcac ctgtatgttg cagattaacg ccggatccta agcttaatta gctgagcttg
     2641 gactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     2701 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     2761 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     2821 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     2881 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     2941 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     3001 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     3061 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     3121 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     3181 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     3241 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     3301 ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca
     3361 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
     3421 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
     3481 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
     3541 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
     3601 aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
     3661 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata
     3721 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
     3781 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
     3841 ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
     3901 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
     3961 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
     4021 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     4081 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc
     4141 gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg
     4201 ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     4261 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     4321 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     4381 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     4441 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4501 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4561 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4621 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     4681 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     4741 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     4801 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     4861 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     4921 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     4981 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     5041 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     5101 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     5161 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     5221 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     5281 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     5341 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     5401 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     5461 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     5521 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     5581 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     5641 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     5701 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     5761 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     5821 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     5881 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     5941 gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     6001 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     6061 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     6121 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     6181 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     6241 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     6301 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     6361 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     6421 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     6481 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     6541 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact
     6601 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     6661 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     6721 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     6781 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     6841 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     6901 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     6961 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     7021 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     7081 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     7141 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     7201 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     7261 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     7321 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     7381 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     7441 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     7501 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     7561 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     7621 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     7681 ccagcaaccg cacctgtggc gccggtgatg ccggc
//
