LOCUS       sequence-321653-        7485 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 7485)
  TITLE     Simon lab plasmids
REFERENCE   2  (bases 1 to 7485)
  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..7485
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     922..941
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(961..1038)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1244..1263
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1347..1366
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1347..1365
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1366..1390
                     /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             1405..1427
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1446..1463
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1473..1490
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3696..3713
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3766..3784)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3780..3827
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3864..4319
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3951..3970)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4161..4182
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4411..5226)
                     /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
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLKFHLMLDEFF"
     primer_bind     5134..5153
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5218..5237
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5348..5936
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5837..5856
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6090..6107
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6122..6261
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6187..6206)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6347..6369
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6363..6554)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7363..7485,1..960))
                     /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"
ORIGIN
        1 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
       61 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
      121 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
      181 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
      241 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
      301 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
      361 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
      421 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
      481 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
      541 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
      601 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
      661 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
      721 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
      781 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
      841 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
      901 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
      961 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     1021 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     1081 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     1141 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     1201 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     1261 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     1321 ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
     1381 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat accatgggca
     1441 gcagccatca tcatcatcat cacagcagcg gcctggtgcc gcgcggcagc catatgaagg
     1501 agtcgacatt ggaagccatc ggttatattt gccaagatat agacccagag cagctacaag
     1561 ataaatccaa tgagattctg actgccataa tccaggggat gaggaaagaa gagcctagta
     1621 ataatgtgaa gctagctgct acgaatgcac tcctgaactc attggagttc accaaagcaa
     1681 actttgataa agagtctgaa aggcacttta ttatgcaggt ggtctgtgaa gccacacagt
     1741 gtccagatac gagggtacga gtggctgctt tacagaatct ggtgaagata atgtccttat
     1801 attatcagta catggagaca tatatgggtc ctgctctttt tgcaatcaca atcgaagcaa
     1861 tgaaaagtga cattgatgag gtggctttac aagggataga attctggtcc aatgtctgtg
     1921 atgaggaaat ggatttggcc attgaagctt cagaggcagc agaacaagga cggccccctg
     1981 agcacaccag caagttttat gcgaagggag cactacagta tctggttcca atcctcacac
     2041 agacactaac taaacaggac gaaaatgatg atgacgatga ctggaacccc tgcaaagcag
     2101 caggggtgtg cctcatgctt ctggccacct gctgtgaaga tgacattgtc ccacatgtcc
     2161 tccccttcat taaagaacac atcaagaacc cagattggcg gtaccgggat gcagcagtga
     2221 tggcttttgg ttgtatcttg gaaggaccag agcccagtca gctcaaacca ctagttatac
     2281 aggctatgcc caccctaata gaattaatga aagaccccag tgtagttgtt cgagatacag
     2341 ctgcatggac tgtaggcaga atttgtgagc tgcttcctga agctgccatc aatgatgtct
     2401 acttggctcc cctgctacag tgtctgattg agggtctcag tgctgaaccc agagtggctt
     2461 caaatgtgtg ctgggctttc tccagtctgg ctgaagctgc ttatgaagct gcagacgttg
     2521 ctgatgatca ggaagaacca gctacttact gcttatcttc ttcatttgaa ctcatagttc
     2581 agaagctcct agagactaca gacagacctg atggacacca gaacaacctg aggagttctg
     2641 catatgaatc tctgatggaa attgtgaaaa acagtgccaa ggattgttat cctgctgtcc
     2701 agaaaacgac tttggtcatc atggaacgac tgcaacaggt tcttcagatg gagtcacata
     2761 tccagagcac atccgataga atccagttca atgaccttca gtctttactc tgtgcaactc
     2821 ttcagaatgt tcttcggaaa gtgcaacatc aagatgcttt gcagatctct gatgtggtta
     2881 tggcctccct gttaaggatg ttccaaagca cagctgggtc tgggggagta caagaggatg
     2941 ccctgatggc agttagcaca ctggtggaag tgttgggtgg tgaattcctc aagtacatgg
     3001 aggcctttaa acccttcctg ggcattggat taaaaaatta tgctgaatac caggtttgtt
     3061 tggcagctgt gggcttagtg ggagacttgt gccgtgccct gcaatccaac atcatacctt
     3121 tctgtgacga ggtgatgcag ctgcttctgg aaaatttggg gaatgagaac gtccacaggt
     3181 ctgtgaagcc gcagattctg tcagtgtttg gtgatattgc ccttgctatt ggaggagagt
     3241 ttaaaaaata cttagaggtt gtattgaata ctcttcagca ggcctcccaa gcccaggtgg
     3301 acaagtcaga ctatgacatg gtggattatc tgaatgagct aagggaaagc tgcttggaag
     3361 cctatactgg aatcgtccag ggattaaagg gggatcagga gaacgtacac ccggatgtga
     3421 tgctggtaca acccagagta gaatttattc tgtctttcat tgaccacatt gctggagatg
     3481 aggatcacac agatggagta gtagcttgtg ctgctggact aataggggac ttatgtacag
     3541 catttgggaa ggatgtactg aaattagtag aagctaggcc aatgatccat gaattgttaa
     3601 ctgaagggcg gagatcgaag actaacaaag caaaaaccct tgctacatgg gcaacaaaag
     3661 aactgaggaa actgaagaac caagcttgac tcgagcacca ccaccaccac cactgagatc
     3721 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
     3781 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     3841 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     3901 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     3961 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     4021 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     4081 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     4141 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     4201 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     4261 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     4321 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     4381 tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa
     4441 cttcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa
     4501 tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc
     4561 gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt
     4621 atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg
     4681 catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc
     4741 atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct
     4801 gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc
     4861 atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc
     4921 ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt
     4981 cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt
     5041 ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa
     5101 tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa
     5161 atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg
     5221 gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca
     5281 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     5341 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     5401 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     5461 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     5521 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     5581 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     5641 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     5701 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     5761 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     5821 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     5881 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     5941 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     6001 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     6061 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     6121 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac
     6181 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     6241 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     6301 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     6361 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     6421 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     6481 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     6541 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     6601 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     6661 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     6721 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     6781 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     6841 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     6901 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     6961 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt
     7021 ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca
     7081 gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc
     7141 atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc
     7201 tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc
     7261 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc
     7321 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag
     7381 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     7441 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagac
//
