LOCUS       sequence-316744-        7489 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of His-tagged KPNB1 in the pET28 protein 
            expression backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7489)
  TITLE     Simon lab plasmids
REFERENCE   2  (bases 1 to 7489)
  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"
COMMENT     Sequence Label: pET28-KPNB1
FEATURES             Location/Qualifiers
     source          1..7489
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     45..67
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(61..252)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1061..2143)
                     /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     2105..2124
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2144..2221)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2427..2446
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2530..2549
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2530..2548
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2549..2573
                     /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             2588..2610
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2629..2646
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2656..2673
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             4879..4896
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4949..4967)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4963..5010
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5047..5502
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5134..5153)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5344..5365
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5595..6410)
                     /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(5595..6410)
                     /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     6318..6337
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6402..6421
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6532..7120
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7021..7040
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7274..7291
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7306..7448
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7374..7393)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
       61 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
      121 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
      181 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
      241 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
      301 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
      361 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
      421 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
      481 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
      541 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
      601 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
      661 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
      721 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
      781 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
      841 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
      901 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
      961 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     1021 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     1081 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     1141 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     1201 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     1261 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     1321 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
     1381 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     1441 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     1501 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     1561 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     1621 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     1681 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     1741 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     1801 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     1861 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     1921 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     1981 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     2041 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     2101 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     2161 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     2221 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     2281 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     2341 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     2401 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     2461 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     2521 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
     2581 aaataatttt gtttaacttt aagaaggaga tataccatgg gcagcagcca tcatcatcat
     2641 catcacagca gcggcctggt gccgcgcggc agccatatga aggagtcgac attggaagcc
     2701 atcggttata tttgccaaga tatagaccca gagcagctac aagataaatc caatgagatt
     2761 ctgactgcca taatccaggg gatgaggaaa gaagagccta gtaataatgt gaagctagct
     2821 gctacgaatg cactcctgaa ctcattggag ttcaccaaag caaactttga taaagagtct
     2881 gaaaggcact ttattatgca ggtggtctgt gaagccacac agtgtccaga tacgagggta
     2941 cgagtggctg ctttacagaa tctggtgaag ataatgtcct tatattatca gtacatggag
     3001 acatatatgg gtcctgctct ttttgcaatc acaatcgaag caatgaaaag tgacattgat
     3061 gaggtggctt tacaagggat agaattctgg tccaatgtct gtgatgagga aatggatttg
     3121 gccattgaag cttcagaggc agcagaacaa ggacggcccc ctgagcacac cagcaagttt
     3181 tatgcgaagg gagcactaca gtatctggtt ccaatcctca cacagacact aactaaacag
     3241 gacgaaaatg atgatgacga tgactggaac ccctgcaaag cagcaggggt gtgcctcatg
     3301 cttctggcca cctgctgtga agatgacatt gtcccacatg tcctcccctt cattaaagaa
     3361 cacatcaaga acccagattg gcggtaccgg gatgcagcag tgatggcttt tggttgtatc
     3421 ttggaaggac cagagcccag tcagctcaaa ccactagtta tacaggctat gcccacccta
     3481 atagaattaa tgaaagaccc cagtgtagtt gttcgagata cagctgcatg gactgtaggc
     3541 agaatttgtg agctgcttcc tgaagctgcc atcaatgatg tctacttggc tcccctgcta
     3601 cagtgtctga ttgagggtct cagtgctgaa cccagagtgg cttcaaatgt gtgctgggct
     3661 ttctccagtc tggctgaagc tgcttatgaa gctgcagacg ttgctgatga tcaggaagaa
     3721 ccagctactt actgcttatc ttcttcattt gaactcatag ttcagaagct cctagagact
     3781 acagacagac ctgatggaca ccagaacaac ctgaggagtt ctgcatatga atctctgatg
     3841 gaaattgtga aaaacagtgc caaggattgt tatcctgctg tccagaaaac gactttggtc
     3901 atcatggaac gactgcaaca ggttcttcag atggagtcac atatccagag cacatccgat
     3961 agaatccagt tcaatgacct tcagtcttta ctctgtgcaa ctcttcagaa tgttcttcgg
     4021 aaagtgcaac atcaagatgc tttgcagatc tctgatgtgg ttatggcctc cctgttaagg
     4081 atgttccaaa gcacagctgg gtctggggga gtacaagagg atgccctgat ggcagttagc
     4141 acactggtgg aagtgttggg tggtgaattc ctcaagtaca tggaggcctt taaacccttc
     4201 ctgggcattg gattaaaaaa ttatgctgaa taccaggttt gtttggcagc tgtgggctta
     4261 gtgggagact tgtgccgtgc cctgcaatcc aacatcatac ctttctgtga cgaggtgatg
     4321 cagctgcttc tggaaaattt ggggaatgag aacgtccaca ggtctgtgaa gccgcagatt
     4381 ctgtcagtgt ttggtgatat tgcccttgct attggaggag agtttaaaaa atacttagag
     4441 gttgtattga atactcttca gcaggcctcc caagcccagg tggacaagtc agactatgac
     4501 atggtggatt atctgaatga gctaagggaa agctgcttgg aagcctatac tggaatcgtc
     4561 cagggattaa agggggatca ggagaacgta cacccggatg tgatgctggt acaacccaga
     4621 gtagaattta ttctgtcttt cattgaccac attgctggag atgaggatca cacagatgga
     4681 gtagtagctt gtgctgctgg actaataggg gacttatgta cagcatttgg gaaggatgta
     4741 ctgaaattag tagaagctag gccaatgatc catgaattgt taactgaagg gcggagatcg
     4801 aagactaaca aagcaaaaac ccttgctaca tgggcaacaa aagaactgag gaaactgaag
     4861 aaccaagctt gactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc
     4921 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     4981 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
     5041 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     5101 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     5161 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     5221 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     5281 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     5341 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     5401 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     5461 aatttaacgc gaattttaac aaaatattaa cgtttacaat ttcaggtggc acttttcggg
     5521 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     5581 tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata
     5641 tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca
     5701 ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca
     5761 acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca
     5821 ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact
     5881 tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta
     5941 ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta
     6001 caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca
     6061 cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg
     6121 agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat
     6181 tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg
     6241 ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca
     6301 cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg
     6361 gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt
     6421 gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga
     6481 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     6541 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     6601 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     6661 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     6721 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     6781 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     6841 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     6901 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     6961 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     7021 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     7081 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     7141 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     7201 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     7261 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     7321 tctccttacg catctgtgcg gtatttcaca ccgcatatat ggtgcactct cagtacaatc
     7381 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     7441 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggc
//
