LOCUS       sequence-429334-        5385 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of the His-tagged peptide PunyEEER..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5385)
  AUTHORS   Suyama A, Devlin KL, Macias-Contreras M, Doh JK, Shinde U, Beatty KE
  TITLE     Orthogonal Versatile Interacting Peptide Tags for Imaging Cellular 
            Proteins.
  JOURNAL   Biochemistry. 2023 Jun 6;62(11):1735-1743. doi: 
            10.1021/acs.biochem.2c00712. Epub 2023 May 11.
  PUBMED    37167569
REFERENCE   2  (bases 1 to 5385)
  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: 
            "10.1021/acs.biochem.2c00712"; journalName: "Biochemistry"; date: 
            "2023-06-6- 6"; volume: "62"; issue: "11"; pages: "1735-1743"
COMMENT     Sequence Label: pET28b-PunyEEER
FEATURES             Location/Qualifiers
     source          1..5385
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1411..1602
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1596..1618)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1704..1843
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1759..1778
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1858..1875)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2029..2617)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2109..2128)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2728..2747)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2739..3554
                     /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             2739..3554
                     /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     complement(2812..2831)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3646..4101)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3783..3804)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3995..4014
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4138..4185)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4181..4199
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4252..4269)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             4438..4460
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4475..4499
                     /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)."
     primer_bind     complement(4499..4518)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4500..4518)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4602..4621)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4827..4904
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(4905..5385,1..602)
                     /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(4924..4943)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc
       61 aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct
      121 ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga
      181 gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg
      241 cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg
      301 ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa gacagctcat
      361 gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg
      421 tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg
      481 tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg
      541 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
      601 gagcgcaacg caattaatgt aagttagctc actcattagg caccgggatc tcgaccgatg
      661 cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc
      721 gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc
      781 tgggtcattt tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt
      841 gcggtattcg gaatcttgca cgccctcgct caagccttcg tcactggtcc cgccaccaaa
      901 cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg ccccacgggt gcgcatgatc
      961 gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat
     1021 gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga
     1081 gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca
     1141 gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga
     1201 acacctacat ctgtattaac gaagcgctgg cattgaccct gagtgatttt tctctggtcc
     1261 cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca
     1321 tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca tcggtatcat tacccccatg
     1381 aacagaaatc ccccttacac ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac
     1441 atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac
     1501 gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc
     1561 agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     1621 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     1681 gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg
     1741 tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac cattgcggtg
     1801 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc
     1861 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     1921 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     1981 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     2041 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     2101 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     2161 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     2221 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     2281 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     2341 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     2401 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     2461 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     2521 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     2581 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     2641 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgaacaataa
     2701 aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa
     2761 cgtcttgctc taggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat
     2821 gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg
     2881 atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg
     2941 agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta
     3001 tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa acagcattcc
     3061 aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc
     3121 tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc
     3181 gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg
     3241 acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat
     3301 tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg
     3361 aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg
     3421 atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt
     3481 ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg
     3541 atgagttttt ctaagaatta attcatgagc ggatacatat ttgaatgtat ttagaaaaat
     3601 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta
     3661 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     3721 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     3781 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     3841 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     3901 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     3961 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     4021 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     4081 atgcgccgct acagggcgcg tcccattcgc caatccggat atagttcctc ctttcagcaa
     4141 aaaacccctc aagacccgtt tagaggcccc aaggggttat gctagttatt gctcagcggt
     4201 ggcagcagcc aactcagctt cctttcgggc tttgttagca gccggatctc agtggtggtg
     4261 gtggtggtgc tcgagaccgt aaacccacgg gtagcacaga ccccaggaac ccagcggacc
     4321 gtaacggtta cggagacgct ggttacgctg ttccaggcgc tgaacttcgg tttccagcgc
     4381 ggtaacttca cgttcgagga acgcattttc gatttccaga gaagaaccca tggtatatct
     4441 ccttcttaaa gttaaacaaa attatttcta gaggggaatt gttatccgct cacaattccc
     4501 ctatagtgag tcgtattaat ttcgcgggat cgagatctcg atcctctacg ccggacgcat
     4561 cgtggccggc atcaccggcg ccacaggtgc ggttgctggc gcctatatcg ccgacatcac
     4621 cgatggggaa gatcgggctc gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat
     4681 ggtggcaggc cccgtggccg ggggactgtt gggcgccatc tccttgcatg caccattcct
     4741 tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa tgcaggagtc
     4801 gcataaggga gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta
     4861 tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt
     4921 tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc
     4981 aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga
     5041 attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg
     5101 ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc
     5161 gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag
     5221 cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact
     5281 atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt
     5341 tatttcttga tgtctctgac cagacaccca tcaacagtat tattt
//
