LOCUS       sequence-387132-        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"
     primer_bind     31..53
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(47..238)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1047..2129)
                     /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     2091..2110
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2130..2207)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2413..2432
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2516..2535
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2516..2534
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2535..2559
                     /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             2574..2596
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2607..2624
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2649..2669
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             3416..3433
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3486..3504)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3500..3547
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3584..4039
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3671..3690)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3881..3902
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4131..4946)
                     /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(4131..4946)
                     /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     4854..4873
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4938..4957
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5068..5656
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5557..5576
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5810..5827
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5842..5981
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5907..5926)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
       61 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
      121 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
      181 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
      241 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
      301 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
      361 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
      421 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
      481 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
      541 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
      601 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
      661 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg
      721 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
      781 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
      841 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
      901 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
      961 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     1021 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     1081 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     1141 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     1201 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     1261 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     1321 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     1381 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     1441 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     1501 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     1561 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     1621 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     1681 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     1741 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     1801 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     1861 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     1921 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     1981 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     2041 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     2101 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     2161 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     2221 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     2281 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     2341 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     2401 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     2461 gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata
     2521 cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt
     2581 aactttaaga aggagatata catatgcacc atcatcatca tcattcttct ggtgtagatc
     2641 tgggtaccga gaacctgtac ttccaatcca tggatctcct tgaacttaca gaggacatgg
     2701 aaaaggaaat cagtaatgcc ctaggccatg gcccacagga tgaaatccta agtagtgctt
     2761 tcaaattgcg aattactcga ggagatattc agacattaaa gaactatcac tggctcaatg
     2821 atgaagtcat taatttttac atgaatcttc tggtggaaag aaataaaaag caaggctatc
     2881 cagcacttca tgtattcagt actttcttct atcctaaatt aaagtctggg ggttaccaag
     2941 cagtgaaacg atggaccaaa ggggtaaatc tctttgaaca agaaattatt ctggtgccta
     3001 ttcatcggaa ggtacattgg agcctggtgg tgattgacct aagaaaaaag tgtcttaaat
     3061 atctggattc tatgggacaa aagggccaca ggatctgtga gattctcctt cagtatttac
     3121 aggatgaaag taagaccaaa agaaatagtg atctgaatct tttagagtgg acccatcaca
     3181 gcatgaaacc acacgagatt cctcaacagc tgaatgggag tgattgtgga atgtttactt
     3241 gtaaatatgc agattatatt tctagggaca aacctatcac atttactcag caccagatgc
     3301 ctctcttccg gaagaagatg gtgtgggaaa tccttcatca gcagttgctg taacagtaaa
     3361 ggtggatacg gatccgaatt cgagctccgt cgacaagctt gcggccgcac tcgagcacca
     3421 ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     3481 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     3541 ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc
     3601 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3661 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3721 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3781 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     3841 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     3901 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     3961 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     4021 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     4081 gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac
     4141 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt
     4201 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca
     4261 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc
     4321 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt
     4381 gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc
     4441 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg
     4501 agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg
     4561 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat
     4621 acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta
     4681 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc
     4741 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc
     4801 gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga
     4861 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa
     4921 gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac
     4981 agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     5041 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     5101 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     5161 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     5221 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     5281 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     5341 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     5401 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     5461 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     5521 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     5581 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     5641 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     5701 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     5761 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     5821 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     5881 ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     5941 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     6001 ccgctgacgc gccctgacgg gcttgtctgc tcccgg
//
