LOCUS       sequence-319497-        6078 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 6xHis-tagged Ku from Mycobacterium tuberculosis under T7 
            promoter for purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6078)
  AUTHORS   Yim SS, Johns NI, Noireaux V, Wang HH
  TITLE     Protecting Linear DNA Templates in Cell-Free Expression Systems from
            Diverse Bacteria.
  JOURNAL   ACS Synth Biol. 2020 Sep 29. doi: 10.1021/acssynbio.0c00277.
  PUBMED    32926785
REFERENCE   2  (bases 1 to 6078)
  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. 2020 Sep 29. doi: 10.1021/acssynbio.0c00277."
COMMENT     Sequence Label: pET28c-Ku
FEATURES             Location/Qualifiers
     source          1..6078
                     /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"
     misc_RNA        23..72
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     RBS             103..114
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             124..141
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1013..1031)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1027..1074
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1111..1566
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1198..1217)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1408..1429
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1658..2473)
                     /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(1658..2473)
                     /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     2381..2400
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2465..2484
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2595..3183
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3084..3103
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3337..3354
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3369..3508
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3434..3453)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3594..3616
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3610..3801)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4610..5692)
                     /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
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGGWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5654..5673
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5693..5770)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5976..5995
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg agctgtcacc ggatgtgctt tccggtctga tgagtccgtg
       61 aggacgaaac agcctctaca aataattttg tttaatctag agaaagagga gaaatactag
      121 atgcatcacc atcaccatca tcgagccatt tggacgggtt cgatcgcctt cgggctggtg
      181 aacgtgccgg tcaaggtgta cagcgctacc gcagaccacg acatcaggtt ccaccaggtg
      241 cacgccaagg acaacggacg catccggtac aagcgcgtct gcgaggcgtg tggcgaggtg
      301 gtcgactacc gcgatcttgc ccgggcctac gagtccggcg acggccaaat ggtggcgatc
      361 accgacgacg acatcgccag cttgcctgaa gaacgcagcc gggagatcga ggtgttggag
      421 ttcgtccccg ccgccgacgt ggacccgatg atgttcgacc gcagctactt tttggagcct
      481 gattcgaagt cgtcgaaatc gtatgtgctg ctggctaaga cactcgccga gaccgaccgg
      541 atggcgatcg tgcatttcac gctgcgcaac aagaccaggc tggcggcgtt gcgcgtcaag
      601 gatttcggca agcgagaggt gatgatggtg cacacgttgc tgtggcccga tgagatccgc
      661 gaccccgact tcccggtgct ggaccagaag gtggagatca aacccgcgga actcaagatg
      721 gccggccagg tggtggactc gatggccgac gacttcaatc cggaccgcta ccacgacacc
      781 taccaggagc agttacagga gctgatcgac accaaactcg aaggtgggca ggcatttacc
      841 gccgaggacc aaccgaggtt gctggacgag cccgaagacg tctccgacct gctcgccaag
      901 ctggaggcca gcgtgaaggc gcgctcgaag gccaactcaa acgtcccaac gcctccgtga
      961 tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
     1021 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     1081 ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
     1141 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     1201 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     1261 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     1321 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     1381 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     1441 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     1501 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     1561 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     1621 aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca
     1681 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     1741 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     1801 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     1861 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
     1921 gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     1981 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
     2041 gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     2101 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     2161 ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     2221 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
     2281 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
     2341 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     2401 catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt
     2461 gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc
     2521 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     2581 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     2641 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     2701 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     2761 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     2821 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     2881 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     2941 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     3001 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     3061 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     3121 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     3181 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     3241 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     3301 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     3361 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat
     3421 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     3481 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     3541 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     3601 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     3661 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     3721 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     3781 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
     3841 aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc
     3901 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
     3961 aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg
     4021 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
     4081 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
     4141 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
     4201 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg
     4261 cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
     4321 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
     4381 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
     4441 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
     4501 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     4561 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     4621 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     4681 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     4741 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     4801 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     4861 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
     4921 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     4981 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagccgc
     5041 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     5101 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     5161 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     5221 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     5281 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     5341 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     5401 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     5461 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     5521 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     5581 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     5641 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     5701 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     5761 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc
     5821 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg
     5881 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
     5941 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
     6001 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag
     6061 atctcgatcc cgcgaaat
//
