LOCUS       sequence-462321-        6402 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression vector expressing yeast S. cerevisiae rai1, no 
            tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6402)
  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 6402)
  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: pZH82 pET28a-Rai1
FEATURES             Location/Qualifiers
     source          1..6402
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     95..114
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     198..217
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        198..216
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    217..241
                     /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             256..278
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1464..1481
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1534..1552)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1548..1595
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1632..2087
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1719..1738)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1929..1950
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2179..2994)
                     /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(2179..2994)
                     /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     2902..2921
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2986..3005
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3116..3704
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3605..3624
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3858..3875
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3890..4029
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3955..3974)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4115..4137
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4131..4322)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5131..6213)
                     /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     6175..6194
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6214..6291)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
       61 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
      121 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
      181 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
      241 ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgggt gttagtgcaa
      301 atttgttcgt aaagcaaaga gggtcgacaa cagctctgaa gcagccaaag gaaataggat
      361 tctattcaag aactaaggat gaagaatatt taattagcga tgatacgaat ttgaattact
      421 actatcttcc agacgctgaa ttggacagga agttggatct ttcatcagga tttcagaaat
      481 ttaaggatta ttacaaagat tttgaagaca ggtgttccct gcgtgggcta ttagaaacca
      541 tagaaagctc tgaaagacat aaaggaaaaa aaattaatgc tgatataatt acttttagag
      601 gaatagcacg gaaattaatt tcatgtgcat ttgactctcc atcttttaat acagttgatc
      661 ttcgtatcgt ttcattcaac ggccaacttt ttatcaagga agtgcctgaa gctgtcaatg
      721 cagctaaggc ttcctctgca actgaagcag ggcgtaatat aaatcaagat ctgaatgtat
      781 ttacagggta caagtttgaa actttggcta ctctttccaa tccgttgcag tatacacccc
      841 gtgaagtgat agaaaaaaga actaaaagga ttgtatcgca tggagatgaa tacattagtg
      901 ttgtgagaac tggtgtaggt aactgcaagt taattcttgg tgctgaggtg gattgtatat
      961 ttgactttaa agaaaacggt agagacaatt taaaacatta tgccgaattg aaatgtacgc
     1021 agcaagttgc gaacatatcg gatacgcata agtttgagcg taaactgttc cggacctggc
     1081 tgcaatgctt cttagttggg atcccaagga tcatatatgg ttttaaagac gaccattatg
     1141 tattaaaaac tgtggaagag ttttcaacgg aggaagtacc tgttttatta aagaacaata
     1201 atccacaagt tggaagtgca tgtttagaag ccattaaatg gtatggtctc cttactgagt
     1261 ggcttttgaa aatgatacca agagatgagg atcctcattc tcaaataaga gcatttaagt
     1321 tggttttcga aaataatcac ctaagattat ccgaaattga agaaagtgat gaggaatatt
     1381 caggtttgat tgatggagaa catatattat cgaacgggtt taaggagtgg agaaaatctt
     1441 tgaaataagc ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca
     1501 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     1561 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     1621 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     1681 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     1741 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     1801 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     1861 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     1921 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     1981 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     2041 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     2101 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     2161 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
     2221 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
     2281 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
     2341 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
     2401 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
     2461 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
     2521 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
     2581 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     2641 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     2701 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     2761 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     2821 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     2881 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     2941 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc
     3001 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac
     3061 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3121 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     3181 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3241 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     3301 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     3361 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     3421 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     3481 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     3541 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     3601 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     3661 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     3721 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     3781 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     3841 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     3901 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     3961 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     4021 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     4081 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     4141 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     4201 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     4261 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     4321 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     4381 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     4441 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     4501 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     4561 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     4621 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     4681 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     4741 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg
     4801 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct
     4861 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc
     4921 cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt
     4981 tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg
     5041 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     5101 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     5161 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     5221 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     5281 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     5341 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     5401 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     5461 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     5521 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     5581 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     5641 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     5701 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     5761 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     5821 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     5881 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     5941 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     6001 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     6061 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     6121 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     6181 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     6241 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     6301 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     6361 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat gg
//
