LOCUS       sequence-371359-        6040 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  T7 expression vector for making his6- and HA3-tagged CLIP protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6040)
  AUTHORS   Baek I, Le SN, Jeon J, Chun Y, Reed C, Buratowski S
  TITLE     A set of Saccharomyces cerevisiae integration vectors for 
            fluorescent dye labeling of proteins.
  JOURNAL   G3 (Bethesda). 2022 Aug 9. pii: 6659097. doi: 
            10.1093/g3journal/jkac201.
  PUBMED    35944214
REFERENCE   2  (bases 1 to 6040)
  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: "G3 
            (Bethesda). 2022 Aug 9. pii: 6659097. doi: 
            10.1093/g3journal/jkac201."
COMMENT     Sequence Label: pET28-his6-HA3-CLIP
FEATURES             Location/Qualifiers
     source          1..6040
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      21..476
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(108..127)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     318..339
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(568..1383)
                     /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(568..1383)
                     /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     1291..1310
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1375..1394
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1505..2093
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1994..2013
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2247..2264
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2279..2418
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2344..2363)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2504..2526
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2520..2711)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3520..4602)
                     /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     4564..4583
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4603..4680)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4886..4905
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4989..5008
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4989..5007
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5008..5032
                     /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             5047..5069
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5088..5105
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5115..5132
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             5136..5168
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             5178..5267
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             5298..5843
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylcytosine 
                     derivatives"
                     /label=CLIP-tag(TM)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVE
                     VPAPAAVLGGPEPLIQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISESHLAALVGNPAATAAVNTALDGNPVPILIPCHRVVQGDSDVGPYLGGLA
                     VKEWLLANEGHRLGKPGLG"
     CDS             5893..5910
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5963..5981)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5977..6024
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
       61 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
      121 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
      181 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
      241 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
      301 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
      361 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
      421 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt
      481 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
      541 aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg
      601 caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga
      661 aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat
      721 tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc
      781 aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat
      841 ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc
      901 aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt
      961 aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc
     1021 aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg
     1081 gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg
     1141 aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc
     1201 aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg
     1261 atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc
     1321 agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct
     1381 cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc atgaccaaaa
     1441 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     1501 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     1561 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     1621 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     1681 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     1741 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     1801 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     1861 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     1921 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     1981 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     2041 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     2101 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     2161 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     2221 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     2281 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct
     2341 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     2401 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     2461 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     2521 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
     2581 tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
     2641 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
     2701 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
     2761 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
     2821 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
     2881 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
     2941 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
     3001 ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg
     3061 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg
     3121 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg
     3181 gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg
     3241 acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc
     3301 gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt
     3361 cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc
     3421 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     3481 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     3541 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     3601 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     3661 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     3721 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     3781 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
     3841 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     3901 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     3961 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     4021 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     4081 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     4141 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     4201 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     4261 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     4321 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     4381 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     4441 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     4501 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     4561 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     4621 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     4681 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
     4741 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
     4801 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
     4861 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
     4921 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc
     4981 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
     5041 aataattttg tttaacttta agaaggagat ataccatggg cagcagccat catcatcatc
     5101 atcacagcag cggcctggtg ccgcgcggca gccatatggc tagcatgact ggtggacagc
     5161 aaatgggtcg cggatcctac ccatacgatg ttcctgacta tgcgggctat ccctatgacg
     5221 tcccggacta tgcaggatcc tatccatatg acgttccaga ttacgctgct cagtgcggtt
     5281 caggtggatc tggttctatg gacaaagact gcgaaatgaa gcgcaccacc ctggatagcc
     5341 ctctgggcaa gctggaactg tctgggtgcg aacagggcct gcaccgtatc atcttcctgg
     5401 gcaaaggaac atctgccgcc gacgccgtgg aagtgcctgc cccagccgcc gtgctgggcg
     5461 gaccagagcc actgatccag gccaccgcct ggctcaacgc ctactttcac cagcctgagg
     5521 ccatcgagga gttccctgtg ccagccctgc accacccagt gttccagcag gagagcttta
     5581 cccgccaggt gctgtggaaa ctgctgaaag tggtgaagtt cggagaggtc atcagcgaga
     5641 gccacctggc cgccctggtg ggcaatcccg ccgccaccgc cgccgtgaac accgccctgg
     5701 acggaaatcc cgtgcccatt ctgatcccct gccaccgggt ggtgcagggc gacagcgacg
     5761 tggggcccta cctgggcggg ctcgccgtga aagagtggct gctggccaac gagggccaca
     5821 gactgggcaa gcctgggctg ggttaaggat ccgaattcga gctccgtcga caagcttgcg
     5881 gccgcactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag
     5941 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     6001 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
//
