LOCUS       sequence-248768-        6354 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of IntNtr-eSrt2A fusion protein (with 
            N-terminal His6-Sumo).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6354)
  TITLE     Muir Lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6354)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pET-His6-Sumo-IntNtr-eSrt2A
FEATURES             Location/Qualifiers
     source          1..6354
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     213..232
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        213..231
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    232..256
                     /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             271..293
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             313..330
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             340..357
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             364..657
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             1427..1444
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1497..1515)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1511..1558
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1595..2050
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1682..1701)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1892..1913
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2142..2957)
                     /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(2142..2957)
                     /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     2865..2884
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2949..2968
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3079..3667
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3568..3587
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3821..3838
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3853..3992
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3918..3937)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4078..4100
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4094..4285)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5094..6176)
                     /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     6138..6157
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6177..6254)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atcgatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
      241 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacat
      301 atgggcagca gccatcatca tcatcatcac ggcagcggcc tggtgccgcg cggcagcgct
      361 agcatgtcgg actcagaagt caatcaagaa gctaagccag aggtcaagcc agaagtcaag
      421 cctgagactc acatcaattt aaaggtgtcc gatggatctt cagagatctt cttcaagatc
      481 aaaaagacca ctcctttaag aaggctgatg gaagcgttcg ctaaaagaca gggtaaggaa
      541 atggactcct taagattctt gtacgacggt attagaattc aagctgatca gacccctgaa
      601 gatttggaca tggaggataa cgatattatt gaggctcaca gagaacagat tggtggtggc
      661 ggcatcctga ccgttgaata tggattcctt cctatcggta agatcgtgga ggaacggatt
      721 gaatgcacag tctatacggt agataaaaat ggctttgtgt atacacaacc tattgctcag
      781 tggcataacc ggggagaaca ggaagttttc gaatactgct tagaagacgg ttcgattatc
      841 cgtgcaacga aagatcacaa atttctgacg accgacggtc agatgttacc gattgatgag
      901 attttcgaac gggggttaga cctgaaacaa gttgatggtt tgccggcaaa accgcagatt
      961 ccgaaagata aaagcaaagt tgcaggttat attgaaattc cggatgcaga tattaaagaa
     1021 ccggtttatc cgggtccggc aacccgtgaa cagctgaatc gtggtgtttg ttttcatgat
     1081 gaaaatgaaa gcctggatga tcagaatatt agcattgcag gtcatacctt tattgatcgt
     1141 ccgaattatc agtttaccaa tctgaaagca gcaaaaccgg gtagcatggt ttattttaaa
     1201 gttggtaatg aaacccgtat ttataaaatg accagcattc gtaaagttca tccgaatgca
     1261 gttggtgttc tggatgaaca ggaaggtaaa gataaacagc tgaccctggt tacctgtgat
     1321 gattataatg aagaaaccgg tgtttgggaa agccgtaaaa tttttgttgc aaccgaagtt
     1381 aaataagaat tcgagctccg tcgacaagct tgcggccgca ctcgagcacc accaccacca
     1441 ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     1501 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     1561 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     1621 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     1681 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     1741 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     1801 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     1861 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     1921 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     1981 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     2041 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     2101 tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc
     2161 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     2221 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     2281 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     2341 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     2401 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     2461 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
     2521 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     2581 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     2641 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     2701 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     2761 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     2821 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     2881 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc
     2941 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     3001 gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     3061 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     3121 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     3181 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     3241 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     3301 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     3361 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     3421 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     3481 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     3541 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     3601 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     3661 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     3721 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     3781 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     3841 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     3901 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     3961 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     4021 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     4081 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     4141 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     4201 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     4261 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     4321 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     4381 atgctcggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     4441 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     4501 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     4561 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     4621 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     4681 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     4741 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     4801 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     4861 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     4921 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     4981 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     5041 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     5101 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5161 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     5221 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     5281 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     5341 ctgtcttcgg tatcgtcgta tcccactacc gagatgtctg caccaacgcg cagcccggac
     5401 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     5461 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     5521 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     5581 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     5641 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     5701 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     5761 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     5821 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     5881 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     5941 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     6001 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     6061 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     6121 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     6181 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     6241 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     6301 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catg
//
