LOCUS       sequence-463813-        5641 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression of mutant mouse cGAS protein (I309T).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5641)
  TITLE     A single amino acid difference across species is responsible for 
            cGAS inhibitor potency shifts
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5641)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pRSFDuet-SUMO-m-cGAS(I309T)
FEATURES             Location/Qualifiers
     source          1..5641
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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)."
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             110..127
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             140..433
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     promoter        2026..2044
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2045..2069
                     /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)."
     CDS             2178..2222
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2260..2278)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2274..2321
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2551..3366)
                     /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(2551..3366)
                     /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     3274..3293
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3367..3458)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3474..4223)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(3484..3501)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3724..3743)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4419..5501)
                     /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     5463..5482
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5502..5579)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(5625..5641,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg
      121 cggcagccac atggcaagca tgtcggactc agaagtcaat caagaagcta agccagaggt
      181 caagccagaa gtcaagcctg agactcacat caatttaaag gtgtccgatg gatcttcaga
      241 gatcttcttc aagatcaaaa agaccactcc tttaagaagg ctgatggaag cgttcgctaa
      301 aagacagggt aaggaaatgg actccttaag attcttgtac gacggtatta gaatccaagc
      361 tgatcagacc cctgaagatt tggacatgga ggataacgat attattgagg ctcacagaga
      421 acagattggt ggatctatgg aagatccgcg tagaaggacg acggcgccac gcgctaagaa
      481 gccgtccgcg aagcgcgccc cgacgcagcc cagcaggacc agggcccacg cggaaagctg
      541 cggcccgcaa aggggggctc gatcgcggcg ggcggagcgt gacggggaca ccacggagaa
      601 gccacgtgcc ccagggcccc gagtgcatcc agcaagggcc actgagctca ccaaagatgc
      661 acagccctcg gccatggacg cggcaggagc caccgcgcgg cctgccgtcc gggtgcccca
      721 gcagcaggcc atcctggatc cggagctgcc cgccgtacgg gagccccagc cgcccgcgga
      781 tcccgaggcg cggaaagtcg taaggggacc tagccacaga aggggcgcgc gctccaccgg
      841 gcagcccaga gcgccgcgag ggtccaggaa ggaaccggac aagctaaaga aggtgctgga
      901 caaattgaga ttgaaacgca aagatatctc ggaggcggcc gagacggtga ataaagttgt
      961 ggaacgcctg ctgcgcagaa tgcagaaacg ggagtcggag ttcaaaggtg tggagcagct
     1021 gaacactggc agctactatg aacatgtgaa gatttctgct cctaatgaat ttgatgttat
     1081 gtttaaactg gaagtcccca ggattgagct acaagaatat tatgaaacag gtgctttcta
     1141 tcttgtgaaa ttcaaaagaa ttccacgagg aaatccgctg agtcatttct tagaagggga
     1201 agtattatca gctaccaaga tgctgtcaaa gtttaggaaa atcattaaag aagaagttaa
     1261 agaaatcaaa gatatagatg tcagtgtgga gaaggaaaaa ccaggaagcc ctgctgtaac
     1321 acttcttatc aggaaccctg aagaaatctc tgtggatata actctggctt tggagtcaaa
     1381 aggcagctgg cctattagta ccaaagaagg actacctatt caaggctggc tgggcacaaa
     1441 agtgaggacc aatctaagac gagagccgtt ttatctcgta cccaagaatg caaaggatgg
     1501 aaatagtttt caaggagaga cctggcgcct ctctttctct cacactgaaa agtacatttt
     1561 gaataatcac gggatagaga aaacatgctg tgaatcttcc ggagcaaaat gctgcagaaa
     1621 agaatgttta aaattaatga aatacctttt ggaacagttg aaaaaagagt ttcaagagct
     1681 ggatgcattc tgttcctacc atgtgaaaac tgccatcttt cacatgtgga cccaggaccc
     1741 gcaggacagt cagtgggacc ccaggaacct cagctcctgc ttcgataagt tgttagcatt
     1801 ctttcttgag tgcctcagga cagagaaact ggatcattat tttattccaa agttcaatct
     1861 attctctcaa gaactaattg accgaaaaag taaagaattt ctatcgaaga aaattgaata
     1921 tgaaagaaat aatgggtttc caatttttga caagctttga gcggccgcat aatgcttaag
     1981 tcgaacagaa agtaatcgta ttgtacacgg ccgcataatc gaaattaata cgactcacta
     2041 taggggaatt gtgagcggat aacaattccc catcttagta tattagttaa gtataagaag
     2101 gagatataca tatggcagat ctcaattgga tatcggccgg ccacgcgatc gctgacgtcg
     2161 gtaccctcga gtctggtaaa gaaaccgctg ctgcgaaatt tgaacgccag cacatggact
     2221 cgtctactag cgcagcttaa ttaacctagg ctgctgccac cgctgagcaa taactagcat
     2281 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaacct caggcatttg
     2341 agaagcacac ggtcacactg cttccggtag tcaataaacc ggtaaaccag caatagacat
     2401 aagcggctat ttaacgaccc tgccctgaac cgacgacaag ctgacgaccg ggtctccgca
     2461 agtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     2521 tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa
     2581 ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa
     2641 tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc
     2701 gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt
     2761 atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg
     2821 catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc
     2881 atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcggtcgct
     2941 gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc
     3001 atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc
     3061 ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt
     3121 cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt
     3181 ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa
     3241 tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa
     3301 atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg
     3361 gctcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     3421 cggatacata tttgaatgta tttagaaaaa taaacaaata ggcatgcagc gctcttccgc
     3481 ttcctcgctc actgactcgc tacgctcggt cgttcgactg cggcgagcgg tgtcagctca
     3541 ctcaaaagcg gtaatacggt tatccacaga atcaggggat aaagccggaa agaacatgtg
     3601 agcaaaaagc aaagcaccgg aagaagccaa cgccgcaggc gtttttccat aggctccgcc
     3661 cccctgacga gcatcacaaa aatcgacgct caagccagag gtggcgaaac ccgacaggac
     3721 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     3781 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     3841 gctcacgctg ttggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     3901 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     3961 acccggtaag acacgactta tcgccactgg cagcagccat tggtaactga tttagaggac
     4021 tttgtcttga agttatgcac ctgttaaggc taaactgaaa gaacagattt tggtgagtgc
     4081 ggtcctccaa cccacttacc ttggttcaaa gagttggtag ctcagcgaac cttgagaaaa
     4141 ccaccgttgg tagcggtggt ttttctttat ttatgagatg atgaatcaat cggtctatca
     4201 agtcaacgaa cagctattcc gttactctag atttcagtgc aatttatctc ttcaaatgta
     4261 gcacctgaag tcagccccat acgatataag ttgtaattct catgttagtc atgccccgcg
     4321 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     4381 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4441 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4501 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     4561 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     4621 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     4681 tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt aatggcgcgc
     4741 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     4801 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     4861 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     4921 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     4981 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     5041 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     5101 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     5161 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     5221 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     5281 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     5341 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     5401 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     5461 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     5521 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     5581 gggatctcga cgctctccct tatgcgactc ctgcattagg aaattaatac gactcactat
     5641 a
//
