LOCUS       sequence-197670-        7135 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transform BL21 Gold(DE3) to induce the expression of HisTnp 
            hyperactive mutations.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7135)
  AUTHORS   Lai B, Tang Q, Jin W, Hu G, Wangsa D, Cui K, Stanton BZ, Ren G, Ding
            Y, Zhao M, Liu S, Song J, Ried T, Zhao K
  TITLE     Trac-looping measures genome structure and chromatin accessibility.
  JOURNAL   Nat Methods. 2018 Sep;15(9):741-747. doi: 10.1038/s41592-018-0107-y.
            Epub 2018 Aug 27.
  PUBMED    30150754
REFERENCE   2  (bases 1 to 7135)
  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"; doi: 
            "10.1038/s41592-018-0107-y"; journalName: "Nat Methods"; date: 
            "2018-09"; volume: "15"; issue: "9"; pages: "741-747"
COMMENT     Sequence Label: pET15bHis6Tnp
FEATURES             Location/Qualifiers
     source          1..7135
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(16..35)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(17..35)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(119..138)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        344..421
                     /gene="lacI"
                     /label=lacI promoter
     CDS             422..1504
                     /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     complement(441..460)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2816..3007
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3001..3023)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3109..3248
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3164..3183
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3263..3280)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3434..4022)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3514..3533)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4182..4973,4974..5042))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4805..4824
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5043..5147)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5215..5233
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(5260..5265,5266..5282,5283..5288)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(5463..5510)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     5506..5524
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(7027..7044)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(7054..7071)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             7090..7112
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    join(7127..7135,1..16)
                     /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)."
ORIGIN
        1 atccgctcac aattccccta tagtgagtcg tattaatttc gcgggatcga gatctcgatc
       61 ctctacgccg gacgcatcgt ggccggcatc accggcgcca caggtgcggt tgctggcgcc
      121 tatatcgccg acatcaccga tggggaagat cgggctcgcc acttcgggct catgagcgct
      181 tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg gactgttggg cgccatctcc
      241 ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc tcaacctact actgggctgc
      301 ttcctaatgc aggagtcgca taagggagag cgtcgagatc ccggacacca tcgaatggcg
      361 caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa
      421 tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt
      481 ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc
      541 ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca
      601 gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt
      661 cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga
      721 acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag
      781 tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg
      841 cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat
      901 tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca
      961 gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg
     1021 ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg
     1081 gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac
     1141 tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc
     1201 cgggctgcgc gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc
     1261 atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag
     1321 cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc
     1381 cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg
     1441 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     1501 gtgagcgcaa cgcaattaat gtaagttagc tcactcatta ggcaccggga tctcgaccga
     1561 tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg
     1621 tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc
     1681 tctgggtcat tttcggcgag gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc
     1741 ttgcggtatt cggaatcttg cacgccctcg ctcaagcctt cgtcactggt cccgccacca
     1801 aacgtttcgg cgagaagcag gccattatcg ccggcatggc ggccgacgcg ctgggctacg
     1861 tcttgctggc gttcgcgacg cgaggctgga tggccttccc cattatgatt cttctcgctt
     1921 ccggcggcat cgggatgccc gcgttgcagg ccatgctgtc caggcaggta gatgacgacc
     1981 atcagggaca gcttcaagga tcgctcgcgg ctcttaccag cctaacttcg atcattggac
     2041 cgctgatcgt cacggcgatt tatgccgcct cggcgagcac atggaacggg ttggcatgga
     2101 ttgtaggcgc cgccctatac cttgtctgcc tccccgcgtt gcgtcgcggt gcatggagcc
     2161 gggccacctc gacctgaatg gaagccggcg gcacctcgct aacggattca ccactccaag
     2221 aattggagcc aatcaattct tgcggagaac tgtgaatgcg caaaccaacc cttggcagaa
     2281 catatccatc gcgtccgcca tctccagcag ccgcacgcgg cgcatctcgg gcagcgttgg
     2341 gtcctggcca cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc taggctggcg
     2401 gggttgcctt actggttagc agaatgaatc accgatacgc gagcgaacgt gaagcgactg
     2461 ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc
     2521 gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc
     2581 aggatgctgc tggctaccct gtggaacacc tacatctgta ttaacgaagc gctggcattg
     2641 accctgagtg atttttctct ggtcccgccg catccatacc gccagttgtt taccctcaca
     2701 acgttccagt aaccgggcat gttcatcatc agtaacccgt atcgtgagca tcctctctcg
     2761 tttcatcggt atcattaccc ccatgaacag aaatccccct tacacggagg catcagtgac
     2821 caaacaggaa aaaaccgccc ttaacatggc ccgctttatc agaagccaga cattaacgct
     2881 tctggagaaa ctcaacgagc tggacgcgga tgaacaggca gacatctgtg aatcgcttca
     2941 cgaccacgct gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa
     3001 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     3061 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac
     3121 ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt
     3181 gtactgagag tgcaccattg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
     3241 gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     3301 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     3361 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     3421 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     3481 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     3541 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     3601 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     3661 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     3721 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     3781 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     3841 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     3901 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     3961 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4021 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4081 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttgcggccgc
     4141 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     4201 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     4261 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     4321 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     4381 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     4441 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctgca
     4501 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     4561 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     4621 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     4681 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     4741 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca
     4801 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     4861 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     4921 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     4981 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     5041 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     5101 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     5161 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     5221 cgtatcacga ggccctttcg tcttcaagaa ttctcatgtt tgacagctta tcatcgataa
     5281 gctttaatgc ggtagtttat cacagttaaa ttgctaacgc agtcaggcac cgtgtatgaa
     5341 atctaacaat gcgctcatcg tcatcctcgg caccgtcacc ctggatgctg taggcatagg
     5401 cttggttatg ccggtactgc cgggcctctt gcgggatatc cggatatagt tcctcctttc
     5461 agcaaaaaac ccctcaagac ccgtttagag gccccaaggg gttatgctag ttattgctca
     5521 gcggtggcag cagccaactc agcttccttt cgggctttgt tagcagccgg atccttatta
     5581 aaggatcttg atcccctgcg ccatcagatc cttggcggca agaaagccat ccagtttact
     5641 ttgcagggct tcccaacctt cccagagggc gccccagctg gcaattccgg ttcgcttgct
     5701 gtccataaaa ccgcccagtc tagctatcgc catgtaagcc cactgcaagc tacctgcttt
     5761 ctctttgcgc ttgcgttttc ccttgtccag atagcccagt agctgacatt catccggggt
     5821 cagcaccgtt tctgcggact ggctttctac gtgttccgct tcctttagca gcccttgcgc
     5881 cctgagtgct tgcggcggcg tgaagctttc tctgagctgt aacagcctga ccgcaacaaa
     5941 cgagaggatc gagaccatcc gctccagatt atccggctcc tccatgcgtt gcctctcggc
     6001 tcctgctccg gttttccatg ccttatggaa ctcctcgatc cgccagcgat gggtataaat
     6061 gtcgatgacg cgcaaggctt gggctagcga ctcgaccggt tcgctggtca gcaacaacca
     6121 tttcaacggg gtctcaccct tgggcgggtt aatctcctcg gccagcaccg cgttgagcgt
     6181 gatattcccc tgttttagcg tgatgcgccc actgcgcagg ctcaagctcg ccttgcgggc
     6241 tggtcgattt ttacgtttac cgcgtttatc caccacgccc ttttgcggaa tgctgatctg
     6301 atagccaccc aactccggtt ggttcttcag atggtcgtac agatacaacc cagactctac
     6361 gtccttgcgt gggtgcttgg agcgcaccac gaagcgctcg ttatgcgcca gtttgtcctg
     6421 cagataagca tgaatatcgg cttcgcggtc acagaccgca atcacgttgc tcatcatgct
     6481 gcccatgcgt aaccggctag ttgcggccgc tgccagccat ttgccactct ccttttcatc
     6541 cgcatcggca gggtcatccg ggcgcatcca ccactcctga tgcagtaatc ctacggtgcg
     6601 gaatgtggtg gcctcgagca agagaacgga gtgaacccac catccgcggg atttatcctg
     6661 aatagagccc agcttgccaa gctcttcggc gacctggtgg cgataactca aagaggtggt
     6721 gtcctcaatg gccagcagtt cgggaaactc ctgagccaac ttgactgttt gcatggcgcc
     6781 agcctttctg atcgcctcgg cagaaacgtt gggattgcgg ataaatcggt aagcgccttc
     6841 ctgcgcggct ttactaccct ctgatgagat ggttattgat ttaccagaat attttgccaa
     6901 ttgggcggcg acgttaacca agcgggcagt acggcgagga tcacccagcg ccgccgaaga
     6961 gaacacagat ttagcccagt cggccgcacg atgaagagca gaagttatca ttccaccacc
     7021 catatggctg ccgcgcggca ccaggccgct gctgtgatga tgatgatgat ggctgctgcc
     7081 catggtatat ctccttctta aagttaaaca aaattatttc tagaggggaa ttgtt
//
