LOCUS       sequence-462323-        8399 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression vector expressing yeast S. cerevisiae rat1 with
            a C-terminal his tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8399)
  AUTHORS   Han Z, Moore GA, Mitter R, Lopez Martinez D, Wan L, Dirac Svejstrup 
            AB, Rueda DS, Svejstrup JQ
  TITLE     DNA-directed termination of RNA polymerase II transcription.
  JOURNAL   Mol Cell. 2023 Sep 21;83(18):3253-3267.e7. doi: 
            10.1016/j.molcel.2023.08.007. Epub 2023 Sep 7.
  PUBMED    37683646
REFERENCE   2  (bases 1 to 8399)
  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: 
            "10.1016/j.molcel.2023.08.007"; journalName: "Mol Cell"; date: 
            "2023-09-21- 21"; volume: "83"; issue: "18"; pages: "3253-3267.e7"
COMMENT     Sequence Label: pZH81 pET21b Rat1-His
FEATURES             Location/Qualifiers
     source          1..8399
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     98..117
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     201..220
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        201..219
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    220..244
                     /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             259..281
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3331..3348
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3401..3419)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3415..3462
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3499..3954
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3586..3605)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3796..3817
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3980..4084
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4085..4153,4154..4945)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4303..4322)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5116..5704
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5605..5624
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5858..5875
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5890..6029
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5955..5974)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6115..6137
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6131..6322)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7131..8213)
                     /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     8175..8194
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8214..8291)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
       61 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
      121 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
      181 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
      241 ttcccctcta gaaataattt tgtttaactt taagaaggag atatacatat ggctagcatg
      301 ggtgttccgt catttttcag atggctatct cgaaaatatc caaagatcat atccccagta
      361 ttggaagagc aacctcagat agtcgacggt gtcatattgc cgttggatta ttccgcctca
      421 aatccaaacg gtgagttaga caacttgtat ttggatatga atggtattgt tcatccatgt
      481 tcgcacccag aaaacaaacc gcctccagag actgaagatg aaatgctttt ggcagttttt
      541 gaatatacca atcgtgtgtt aaatatggct agaccacgca aggtacttgt tatggctgtt
      601 gatggtgttg ctcctcgtgc caaaatgaac cagcagagag cgcgtagatt taggagtgct
      661 agggatgctc agattgaaaa cgaagccagg gaagaaatta tgagacagcg agaagaagtt
      721 ggtgagataa tcgatgatgc cgtcaggaac aagaaaactt gggattcaaa tgcgatcact
      781 ccaggcaccc catttatgga taagcttgcc gcggccctcc gttattggac agcatttaaa
      841 ctggccactg accctggttg gaaaaacctg caggtgatta tcagtgacgc caccgtccca
      901 ggtgaaggtg agcacaaaat aatgaatttc attaggtctc aaagagctga tcctgaatat
      961 aatcccaaca caacgcactg tatatacggt ttggatgctg atttaatttt tttgggcctt
     1021 gccactcatg aaccacattt taaaattcta agagaagatg tttttgctca agacaatcgg
     1081 aaaagaaata acttaaaaga tactataaat atgactgaag aagaaaaaca atttctccaa
     1141 aaacaaaatt ccgaacaacc tttcttgtgg ctgcacataa atgttttgag ggaatacttg
     1201 tccgctgagt tatgggttcc gggtcttcca tttacatttg atttagaaag ggctatcgac
     1261 gattgggttt tcatgtgctt tttctgtggt aatgactttt taccacattt accatgctta
     1321 gatgtcagag aaaatagtat tgatattctc ttggatattt ggaaggtagt actaccgaaa
     1381 ttgaaaacgt acatgacctg cgacggtgtc ttgaacctcc cttctgttga aacgctattg
     1441 caacaccttg gctcccgtga gggggatatc ttcaaaacta gacatattca agaagccaga
     1501 aagaaagaag cttttgaaag aaggaaagcg cagaaaaata tgtcaaaagg tcaagacagg
     1561 catcctacag tagctacgga gcaactgcaa atgtatgaca cacaaggaaa tttagcaaaa
     1621 ggttcatgga acttgaccac cagtgatatg gtaaggttga aaaaggaact gatgcttgcg
     1681 aacgagggga acgaagaagc aatcgccaaa gttaagcaac agagtgataa gaacaatgaa
     1741 ctgatgaaag atatcagcaa ggaagaaatt gatgatgctg ttagtaaggc taacaaaact
     1801 aattttaacc ttgcagaagt tatgaaacag aaaataataa ataagaaaca ccgtcttgaa
     1861 aaggataatg aagaggaaga aattgctaag gatagcaaaa aagttaagac agagaaagcg
     1921 gaatcagagt gcgatctcga tgcagagatc aaagatgaga ttgtggctga cgtaaacgac
     1981 agggaaaact ctgaaactac agaagtatca agagattcac cagtccacag cacagtgaat
     2041 gtcagcgaag gtcccaaaaa tggggtcttt gatacagatg aatttgtcaa actattcgaa
     2101 cctggttacc acgaaagata ttacacagct aaatttcacg ttactcctca agatattgaa
     2161 cagttaagaa aagatatggt aaaatgctat atcgaagggg tcgcctgggt actaatgtat
     2221 tactatcaag gttgtgcctc ttggaattgg ttctatccgt accattatgc tcctctggca
     2281 acagatttcc atgggttttc tcacttggag ataaagttcg aagaaggtac tccattttta
     2341 ccttacgaac aattgatgag tgttttacca gctgcatctg gccatgcttt gccaaaaatt
     2401 ttccggtctt tgatgtctga acctgactct gaaatcattg atttttatcc tgaagaattt
     2461 cccatagata tgaatggtaa gaagatgtcc tggcaaggca tagcgttgct accattcatc
     2521 gatcaggata gactgttaac agctgttcgt gcacaatatc ctttactttc tgatgctgag
     2581 agggcaagaa atattcgcgg agaacctgtt ttattaataa gtaacaagaa tgctaattat
     2641 gagaggtttt cgaagaaatt atactcaaag gaaaacaaca acaacaatgt tgttgtgaag
     2701 tttcaacact tcaagagtgg tttaagtggt attgtatcaa aggacgttga aggtttcgaa
     2761 ttaaatggga aaattgtatg tccgattcaa ggtgggtcac tgccaaattt atctaccacc
     2821 ttgattttaa agatgtccta tagattaatt ccgttgccat cgagaaataa gtctattata
     2881 ctgaacgggt tcattccgtc agaaccggta ctaacagcat atgaccttga ttccataatg
     2941 tacaagtata ataaccaaaa ttactcccgt cgctggaact ttggtaatga tttgaagcaa
     3001 aatattgttc cagtaggacc aaagggtatt actcagtaca aaccaagaac tggtggctat
     3061 cgagcattct tttattttgc tgagctcagt agaaataatg tccaacccgc ccacaactat
     3121 ggtaggaata gctacaacag tcaacccggc ttcaacaata gcaggtatga tggtggaaac
     3181 aataattaca gacaaaattc aaattacagg aacaataact attccggaaa tagaaatagt
     3241 ggacagtaca gcggcaacag ctactctcgg aataacaagc aaagtcggta tgacaattca
     3301 agagcaaata ggcgtgcggc cgcactcgag caccaccacc accaccactg agatccggct
     3361 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
     3421 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
     3481 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     3541 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     3601 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     3661 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     3721 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     3781 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     3841 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     3901 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     3961 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     4021 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     4081 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     4141 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     4201 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     4261 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     4321 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     4381 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     4441 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     4501 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     4561 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     4621 gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     4681 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     4741 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     4801 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     4861 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     4921 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     4981 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     5041 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     5101 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     5161 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     5221 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     5281 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     5341 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     5401 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     5461 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     5521 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     5581 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     5641 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     5701 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     5761 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     5821 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     5881 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa
     5941 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     6001 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     6061 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     6121 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     6181 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     6241 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     6301 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
     6361 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
     6421 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga
     6481 gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg
     6541 gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc
     6601 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg
     6661 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct
     6721 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc
     6781 gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg
     6841 cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca
     6901 ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg
     6961 ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag
     7021 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     7081 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     7141 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     7201 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
     7261 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
     7321 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
     7381 tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg
     7441 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
     7501 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
     7561 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
     7621 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
     7681 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
     7741 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
     7801 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
     7861 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
     7921 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
     7981 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
     8041 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
     8101 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
     8161 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     8221 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     8281 tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc
     8341 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggaga
//
