LOCUS       sequence-493788-        5365 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty backbone for bacterial expression. 8xHis tag, K in position 2 
            to minimise posttranslational gluconoylation..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5365)
  TITLE     pET28a(+)-8HisG2K
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5365)
  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            "Unpublished"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: pET28a(+)-8HisG2K
FEATURES             Location/Qualifiers
     source          1..5365
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(207..239)
                     /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             complement(243..260)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(270..293)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     RBS             306..328
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    343..367
                     /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(367..386)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(368..386)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(470..489)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        695..772
                     /gene="lacI"
                     /label=lacI promoter
     CDS             773..1855
                     /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(792..811)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2664..2855
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2849..2871)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2957..3096
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3012..3031
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3111..3128)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3282..3870)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3362..3381)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(3981..4000)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3992..4807
                     /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             3992..4807
                     /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(4065..4084)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(4899..5354)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5036..5057)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5248..5267
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattcgga tccgcgaccc atttgctgtc caccagtcat gctagccata
      241 tggctgccgc gcggcaccag gccgctgctg tgatgatgat gatgatggtg gtgtttcatg
      301 gtatatctcc ttcttaaagt taaacaaaat tatttctaga ggggaattgt tatccgctca
      361 caattcccct atagtgagtc gtattaattt cgcgggatcg agatctcgat cctctacgcc
      421 ggacgcatcg tggccggcat caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc
      481 gacatcaccg atggggaaga tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc
      541 gtgggtatgg tggcaggccc cgtggccggg ggactgttgg gcgccatctc cttgcatgca
      601 ccattccttg cggcggcggt gctcaacggc ctcaacctac tactgggctg cttcctaatg
      661 caggagtcgc ataagggaga gcgtcgagat cccggacacc atcgaatggc gcaaaacctt
      721 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc
      781 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
      841 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
      901 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
      961 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
     1021 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
     1081 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
     1141 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt
     1201 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca
     1261 tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
     1321 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa
     1381 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
     1441 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
     1501 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
     1561 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat
     1621 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
     1681 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact
     1741 ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     1801 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     1861 acgcaattaa tgtaagttag ctcactcatt aggcaccggg atctcgaccg atgcccttga
     1921 gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac
     1981 ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca
     2041 ttttcggcga ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat
     2101 tcggaatctt gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg
     2161 gcgagaagca ggccattatc gccggcatgg cggccccacg ggtgcgcatg atcgtgctcc
     2221 tgtcgttgag gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac
     2281 cgatacgcga gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa
     2341 catgaatggt cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct
     2401 gcaccattat gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta
     2461 catctgtatt aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca
     2521 tccataccgc cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag
     2581 taacccgtat cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa
     2641 atccccctta cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc
     2701 gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg
     2761 aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc
     2821 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     2881 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     2941 ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg
     3001 gcttaactat gcggcatcag agcagattgt actgagagtg caccattgcg gtgtgaaata
     3061 ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact
     3121 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     3181 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     3241 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     3301 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     3361 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     3421 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     3481 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     3541 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     3601 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     3661 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     3721 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     3781 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     3841 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     3901 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgaacaa taaaactgtc
     3961 tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcttg
     4021 ctctaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg
     4081 cgataatgtc gggcaatcag gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc
     4141 agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt
     4201 cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac
     4261 tcctgatgat gcatggttac tcaccactgc gatccccggg aaaacagcat tccaggtatt
     4321 agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg
     4381 gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc
     4441 tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg
     4501 taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc cattctcacc
     4561 ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa
     4621 attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc
     4681 catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa
     4741 atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt
     4801 tttctaagaa ttaattcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     4861 taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt
     4921 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     4981 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     5041 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     5101 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     5161 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     5221 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     5281 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     5341 gctacagggc gcgtcccatt cgcca
//
