LOCUS       sequence-240376-        5292 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial 6xHIS tagged protein expression without the addition of 
            any potential phosphorylation sites to the tagged protein..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5292)
  TITLE     Feher Lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5292)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pET28b_NOSER
FEATURES             Location/Qualifiers
     source          1..5292
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     90..109
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        90..108
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    109..133
                     /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             148..170
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             189..206
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             246..263
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(316..334)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      330..377
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      414..869
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(501..520)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     711..732
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(961..1776)
                     /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(961..1776)
                     /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     1684..1703
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1768..1787
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1898..2486
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2387..2406
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2640..2657
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2672..2811
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2737..2756)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2897..2919
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2913..3104)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3913..4995)
                     /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     4957..4976
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4996..5073)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(5279..5292,1..6)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
       61 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
      121 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tataccatgg
      181 gcggcggaca tcatcatcat catcacgaat tcgagctcgt cgacaagctt gcggccgcac
      241 tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg
      301 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
      361 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc
      421 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
      481 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
      541 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
      601 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
      661 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
      721 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
      781 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
      841 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
      901 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc
      961 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
     1021 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     1081 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     1141 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     1201 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca
     1261 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     1321 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
     1381 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     1441 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     1501 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     1561 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     1621 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     1681 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     1741 cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
     1801 gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact
     1861 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     1921 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     1981 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     2041 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     2101 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     2161 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     2221 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     2281 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     2341 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     2401 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     2461 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     2521 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     2581 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     2641 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     2701 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     2761 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     2821 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     2881 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     2941 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     3001 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     3061 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     3121 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     3181 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     3241 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     3301 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     3361 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     3421 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     3481 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     3541 aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc
     3601 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag
     3661 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg
     3721 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     3781 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     3841 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     3901 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     3961 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     4021 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     4081 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     4141 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
     4201 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     4261 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     4321 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     4381 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     4441 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     4501 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     4561 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     4621 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     4681 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     4741 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     4801 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     4861 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     4921 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     4981 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     5041 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     5101 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     5161 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     5221 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     5281 tgatgtcggc ga
//
