LOCUS       sequence-463815-        5686 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression of mutant human cGAS protein (T321I).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5686)
  TITLE     A single amino acid difference across species is responsible for 
            cGAS inhibitor potency shifts
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5686)
  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            "Unpublished"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-h-cGAS(T321I)
FEATURES             Location/Qualifiers
     source          1..5686
                     /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        2071..2089
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2090..2114
                     /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             2223..2267
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2305..2323)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2319..2366
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2596..3411)
                     /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(2596..3411)
                     /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     3319..3338
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3412..3503)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3519..4268)
                     /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(3529..3546)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3769..3788)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4464..5546)
                     /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     5508..5527
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5547..5624)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(5670..5686,1..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 ggatctatgc agccttggca cggaaaggcc atgcagagag cttccgaggc
      481 cggagccact gcccccaagg cttccgcacg gaatgccagg ggcgccccga tggatcccaa
      541 cgagtctccg gctgcccccg aggccgccct gcctaaggcg ggaaagttcg gccccgccag
      601 gaagtcggga tcccggcaga aaaagagcgc cccggacacc caggagaggc cgcccgtccg
      661 cgcaactggg gcccgcgcca aaaaggcccc tcagcgcgcc caggacacgc agccgtctga
      721 cgccaccagc gcccctgggg cagaggggct ggagcctcct gcggctcggg agccggctct
      781 ttccagggct ggttcttgcc gccagagggg cgcgcgctgc tccacgaagc caagaccccc
      841 gcccgggccc tgggacgtgc ccagccccgg cctgccggtc tcggccccca ttctcgtacg
      901 gagggatgcg gcgcctgggg cctcgaagct ccgggcggtt ttggagaagt tgaagctcag
      961 ccgcgatgat atctccacgg cggcggggat ggtgaaaggg gttgtggacc acctgctgct
     1021 cagactgaag tgcgactccg cgttcagagg cgtcgggctg ctgaacaccg ggagctacta
     1081 tgagcacgtg aagatttctg cacctaatga atttgatgtc atgtttaaac tggaagtccc
     1141 cagaattcaa ctagaagaat attccaacac tcgtgcatat tactttgtga aatttaaaag
     1201 aaatccgaaa gaaaatcatc tgagtcagtt tttagaaggt gaaatattat cagcttctaa
     1261 gatgctgtca aagtttagga aaatcattaa ggaagaaatt aacgacatta aagatacaga
     1321 tgtcatcatg aagaggaaaa gaggagggag ccctgctgta acacttctta ttagtgaaaa
     1381 aatatctgtg gatataatcc tggctttgga atcaaaaagt agctggcctg ctagcaccca
     1441 agaaggcctg cgcattcaaa actggctttc agcaaaagtt aggaagcaac tacgactaaa
     1501 gccattttac cttgtaccca agcatgcaaa ggaaggaaat ggtttccaag aagaaacatg
     1561 gcggctatcc ttctctcaca tcgaaaagga aattttgaac aatcatggaa aatctaaaac
     1621 gtgctgtgaa aacaaagaag agaaatgttg caggaaagat tgtttaaaac taatgaaata
     1681 ccttttagaa cagctgaaag aaaggtttaa agacaaaaaa catctggata aattctcttc
     1741 ttatcatgtg aaaactgcct tctttcacgt atgtacccag aaccctcaag acagtcagtg
     1801 ggaccgcaaa gacctgggcc tctgctttga taactgcgtg acatactttc ttcagtgcct
     1861 caggacagaa aaacttgaga attattttat tcctgaattc aatctattct ctagcaactt
     1921 aattgacaaa agaagtaaag aatttctgac aaagcaaatt gaatatgaaa gaaacaatga
     1981 gtttccagtt tttgatgaat tttgagcggc cgcataatgc ttaagtcgaa cagaaagtaa
     2041 tcgtattgta cacggccgca taatcgaaat taatacgact cactataggg gaattgtgag
     2101 cggataacaa ttccccatct tagtatatta gttaagtata agaaggagat atacatatgg
     2161 cagatctcaa ttggatatcg gccggccacg cgatcgctga cgtcggtacc ctcgagtctg
     2221 gtaaagaaac cgctgctgcg aaatttgaac gccagcacat ggactcgtct actagcgcag
     2281 cttaattaac ctaggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     2341 ctaaacgggt cttgaggggt tttttgctga aacctcaggc atttgagaag cacacggtca
     2401 cactgcttcc ggtagtcaat aaaccggtaa accagcaata gacataagcg gctatttaac
     2461 gaccctgccc tgaaccgacg acaagctgac gaccgggtct ccgcaagtgg cacttttcgg
     2521 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     2581 ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     2641 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     2701 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     2761 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     2821 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     2881 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     2941 attcattcgt gattgcgcct gagcgagacg aaatacgcgg tcgctgttaa aaggacaatt
     3001 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     3061 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     3121 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     3181 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     3241 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     3301 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     3361 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca tactcttcct
     3421 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     3481 atgtatttag aaaaataaac aaataggcat gcagcgctct tccgcttcct cgctcactga
     3541 ctcgctacgc tcggtcgttc gactgcggcg agcggtgtca gctcactcaa aagcggtaat
     3601 acggttatcc acagaatcag gggataaagc cggaaagaac atgtgagcaa aaagcaaagc
     3661 accggaagaa gccaacgccg caggcgtttt tccataggct ccgcccccct gacgagcatc
     3721 acaaaaatcg acgctcaagc cagaggtggc gaaacccgac aggactataa agataccagg
     3781 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     3841 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgttggt
     3901 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     3961 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4021 acttatcgcc actggcagca gccattggta actgatttag aggactttgt cttgaagtta
     4081 tgcacctgtt aaggctaaac tgaaagaaca gattttggtg agtgcggtcc tccaacccac
     4141 ttaccttggt tcaaagagtt ggtagctcag cgaaccttga gaaaaccacc gttggtagcg
     4201 gtggtttttc tttatttatg agatgatgaa tcaatcggtc tatcaagtca acgaacagct
     4261 attccgttac tctagatttc agtgcaattt atctcttcaa atgtagcacc tgaagtcagc
     4321 cccatacgat ataagttgta attctcatgt tagtcatgcc ccgcgcccac cggaaggagc
     4381 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     4441 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4501 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     4561 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     4621 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     4681 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     4741 gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     4801 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     4861 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     4921 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     4981 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     5041 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     5101 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     5161 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     5221 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     5281 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     5341 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     5401 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     5461 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     5521 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     5581 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     5641 tcccttatgc gactcctgca ttaggaaatt aatacgactc actata
//
