LOCUS       sequence-321641-        6879 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of His-tagged KPNA1 in the pET28 protein 
            expression backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6879)
  TITLE     Simon lab plasmids
REFERENCE   2  (bases 1 to 6879)
  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_KPNA1
FEATURES             Location/Qualifiers
     source          1..6879
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     17..36
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     120..139
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        120..138
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    139..163
                     /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             178..200
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             219..236
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             246..263
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1863..1880
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1933..1951)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1947..1994
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2031..2486
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2118..2137)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2328..2349
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2578..3393)
                     /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     3301..3320
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3385..3404
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3515..4103
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4004..4023
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4257..4274
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4289..4428
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4354..4373)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4514..4536
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4530..4721)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5530..6612)
                     /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     6574..6593
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6613..6690)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
       61 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt
      121 aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt
      181 gtttaacttt aagaaggaga tataccatgg gcagcagcca tcatcatcat catcacagca
      241 gcggcctggt gccgcgcggc agccatatgt ccaccaacga gaatgctaat acaccagctg
      301 cccgtcttca cagattcaag aacaagggaa aagacagtac agaaatgagg cgtcgcagaa
      361 tagaggtcaa tgtggagctg aggaaagcta agaaggatga ccagatgctg aagaggagaa
      421 atgtaagctc atttcctgat gatgctactt ctccgctgca ggaaaaccgc aacaaccagg
      481 gcactgtaaa ttggtctgtt gatgacattg tcaaaggcat aaatagcagc aatgtggaaa
      541 atcagctcca agctactcaa gctgccagga aactactttc cagagaaaaa cagcccccca
      601 tagacaacat aatccgggct ggtttgattc cgaaatttgt gtccttcttg ggcagaactg
      661 attgtagtcc cattcagttt gaatctgctt gggcactcac taacattgct tctgggacat
      721 cagaacaaac caaggctgtg gtagatggag gtgccatccc agcattcatt tctctgttgg
      781 catctcccca tgctcacatc agtgaacaag ctgtctgggc tctaggaaac attgcaggtg
      841 atggctcagt gttccgagac ttggttatta agtacggtgc agttgaccca ctgttggctc
      901 tccttgcagt tcctgatatg tcatctttag catgtggcta cttacgtaat cttacctgga
      961 cactttctaa tctttgccgc aacaagaatc ctgcaccccc gatagatgct gttgagcaga
     1021 ttcttcctac cttagttcgg ctcctgcatc atgatgatcc agaagtgtta gcagatacct
     1081 gctgggctat ttcctacctt actgatggtc caaatgaacg aattggcatg gtggtgaaaa
     1141 caggagttgt gccccaactt gtgaagcttc taggagcttc tgaattgcca attgtgactc
     1201 ctgccctaag agccataggg aatattgtca ctggtacaga tgaacagact caggttgtga
     1261 ttgatgcagg agcactcgcc gtctttccca gcctgctcac caaccccaaa actaacattc
     1321 agaaggaagc tacgtggaca atgtcaaaca tcacagccgg ccgccaggac cagatacagc
     1381 aagttgtgaa tcatggatta gtcccattcc ttgtcagtgt tctctctaag gcagatttta
     1441 agacacaaaa ggaagctgtg tgggccgtga ccaactatac cagtggtgga acagttgaac
     1501 agattgtgta ccttgttcac tgtggcataa tagaaccgtt gatgaacctc ttaactgcaa
     1561 aagataccaa gattattctg gttatcctgg atgccatttc aaatatcttt caggctgctg
     1621 agaaactagg tgaaactgag aaacttagta taatgattga agaatgtgga ggcttagaca
     1681 aaattgaagc tctacaaaac catgaaaatg agtctgtgta taaggcttcg ttaagcttaa
     1741 ttgagaagta tttctctgta gaggaagagg aagatcaaaa cgttgtacca gaaactacct
     1801 ctgaaggcta cactttccaa gttcaggatg gggctcctgg gacctttaac ttttagctcg
     1861 agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt
     1921 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     1981 tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg
     2041 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     2101 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     2161 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     2221 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     2281 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     2341 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     2401 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     2461 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     2521 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta
     2581 gaaaaactca tcgagcatca aatgaaactt caatttattc atatcaggat tatcaatacc
     2641 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag
     2701 gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat
     2761 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga
     2821 atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc
     2881 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc
     2941 ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg
     3001 caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc
     3061 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc
     3121 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag
     3181 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa
     3241 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt
     3301 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct
     3361 agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta
     3421 agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     3481 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     3541 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     3601 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     3661 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     3721 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     3781 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     3841 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     3901 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     3961 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4021 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4081 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4141 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     4201 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     4261 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     4321 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     4381 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     4441 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     4501 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     4561 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     4621 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     4681 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     4741 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     4801 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     4861 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     4921 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     4981 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     5041 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     5101 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     5161 gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc
     5221 tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt
     5281 ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg
     5341 aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc
     5401 ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     5461 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     5521 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     5581 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     5641 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     5701 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     5761 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc
     5821 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     5881 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     5941 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     6001 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     6061 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     6121 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     6181 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     6241 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     6301 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     6361 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     6421 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     6481 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     6541 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     6601 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     6661 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
     6721 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     6781 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     6841 ccacgccgaa acaagcgctc atgagcccga agtggcgag
//
