LOCUS       sequence-392708-        6036 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  protein expression of the His-tagged catalytic domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6036)
  TITLE     Knapp lab plasmids
REFERENCE   2  (bases 1 to 6036)
  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: pNIC28-SENP2
FEATURES             Location/Qualifiers
     source          1..6036
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    41..180
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     96..115
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(195..212)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(366..954)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(446..465)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1065..1084)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1076..1891
                     /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             1076..1891
                     /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(1149..1168)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(1983..2438)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2120..2141)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2332..2351
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(2475..2522)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2518..2536
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2589..2606)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(3353..3373)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             complement(3398..3415)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             3426..3448
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3463..3487
                     /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(3487..3506)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3488..3506)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3590..3609)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3815..3892
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3893..4975
                     /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(3912..3931)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5784..5975
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5969..5991)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac
       61 gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag
      121 agtgcaccat tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
      181 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
      241 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
      301 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
      361 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
      421 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
      481 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
      541 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
      601 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
      661 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
      721 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
      781 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
      841 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
      901 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
      961 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     1021 ttggtcatga acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag
     1081 ccatattcaa cgggaaacgt cttgctctag gccgcgatta aattccaaca tggatgctga
     1141 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg
     1201 attgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc
     1261 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc
     1321 gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc
     1381 cgggaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga
     1441 tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa
     1501 cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga
     1561 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat
     1621 gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga
     1681 taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat
     1741 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc
     1801 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca
     1861 gtttcatttg atgctcgatg agtttttcta agaattaatt catgagcgga tacatatttg
     1921 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     1981 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     2041 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     2101 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     2161 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     2221 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     2281 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     2341 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     2401 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccaa tccggatata
     2461 gttcctcctt tcagcaaaaa acccctcaag acccgtttag aggccccaag gggttatgct
     2521 agttattgct cagcggtggc agcagccaac tcagcttcct ttcgggcttt gttagcagcc
     2581 ggatctcagt ggtggtggtg gtggtgctcg agtgcggccg caagcttgtc gacggagctc
     2641 gaattcggat ccgtatccac ctttactgtt acagcaactg ctgatgaagg atttcccaca
     2701 ccatcttctt ccggaagaga ggcatctggt gctgagtaaa tgtgataggt ttgtccctag
     2761 aaatataatc tgcatattta caagtaaaca ttccacaatc actcccattc agctgttgag
     2821 gaatctcgtg tggtttcatg ctgtgatggg tccactctaa aagattcaga tcactatttc
     2881 ttttggtctt actttcatcc tgtaaatact gaaggagaat ctcacagatc ctgtggccct
     2941 tttgtcccat agaatccaga tatttaagac acttttttct taggtcaatc accaccaggc
     3001 tccaatgtac cttccgatga ataggcacca gaataatttc ttgttcaaag agatttaccc
     3061 ctttggtcca tcgtttcact gcttggtaac ccccagactt taatttagga tagaagaaag
     3121 tactgaatac atgaagtgct ggatagcctt gctttttatt tctttccacc agaagattca
     3181 tgtaaaaatt aatgacttca tcattgagcc agtgatagtt ctttaatgtc tgaatatctc
     3241 ctcgagtaat tcgcaatttg aaagcactac ttaggatttc atcctgtggg ccatggccta
     3301 gggcattact gatttccttt tccatgtcct ctgtaagttc aaggagatcc atggattgga
     3361 agtacaggtt ctcggtaccc agatctacac cagaagaatg atgatgatga tggtgcatat
     3421 gtatatctcc ttcttaaagt taaacaaaat tatttctaga ggggaattgt tatccgctca
     3481 caattcccct atagtgagtc gtattaattt cgcgggatcg agatctcgat cctctacgcc
     3541 ggacgcatcg tggccggcat caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc
     3601 gacatcaccg atggggaaga tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc
     3661 gtgggtatgg tggcaggccc cgtggccggg ggactgttgg gcgccatctc cttgcatgca
     3721 ccattccttg cggcggcggt gctcaacggc ctcaacctac tactgggctg cttcctaatg
     3781 caggagtcgc ataagggaga gcgtcgagat cccggacacc atcgaatggc gcaaaacctt
     3841 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc
     3901 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
     3961 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
     4021 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
     4081 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
     4141 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
     4201 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
     4261 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt
     4321 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca
     4381 tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
     4441 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa
     4501 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
     4561 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
     4621 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
     4681 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat
     4741 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
     4801 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact
     4861 ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     4921 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     4981 acgcaattaa tgtaagttag ctcactcatt aggcaccggg atctcgaccg atgcccttga
     5041 gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac
     5101 ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca
     5161 ttttcggcga ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat
     5221 tcggaatctt gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg
     5281 gcgagaagca ggccattatc gccggcatgg cggccccacg ggtgcgcatg atcgtgctcc
     5341 tgtcgttgag gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac
     5401 cgatacgcga gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa
     5461 catgaatggt cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct
     5521 gcaccattat gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta
     5581 catctgtatt aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca
     5641 tccataccgc cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag
     5701 taacccgtat cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa
     5761 atccccctta cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc
     5821 gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg
     5881 aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc
     5941 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     6001 cagcttgtct gtaagcggat gccgggagca gacaag
//
