LOCUS       sequence-380469-        5856 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses nuclease A-H124L from Staphylococcus aureus, with an A112C
            mutation, fused through a flexible linker to the transmembrane 
            domain of GpA and a His-tag, for fluorescent studies..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5856)
  AUTHORS   Diaz Vazquez G, Cui Q, Senes A
  TITLE     Thermodynamic analysis of the GAS(right) transmembrane motif 
            supports energetic model of dimerization.
  JOURNAL   Biophys J. 2023 Jan 3;122(1):143-155. doi: 
            10.1016/j.bpj.2022.11.018. Epub 2022 Nov 12.
  PUBMED    36371634
REFERENCE   2  (bases 1 to 5856)
  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"; doi: 
            "10.1016/j.bpj.2022.11.018"; journalName: "Biophys J"; date: 
            "2023-01-3- 3"; volume: "122"; issue: "1"; pages: "143-155"
COMMENT     Sequence Label: pSN-A112C-GpA
FEATURES             Location/Qualifiers
     source          1..5856
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    14..38
                     /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             53..75
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             85..531
                     /codon_start=1
                     /product="micrococcal nuclease from Staphylococcus aureus"
                     /label=MNase
                     /translation="ATSTKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLLVDTPETK
                     HPKKGVEKYGPEASAFTKKMVENAKKIEVEFDKGQRTDKYGRGLAYIYADGKMVNEALV
                     RQGLAKVCYVYKPNNTHEQLLRKSEAQAKKEKLNIWSEDNADSGQ"
     CDS             715..732
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(785..803)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      799..846
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      883..1338
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(970..989)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1180..1201
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1430..2245)
                     /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
                     TAFQVLEEYPDSGENIVDVLAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2153..2172
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2237..2256
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2367..2955
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2856..2875
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3109..3126
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3141..3280
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3206..3225)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3366..3388
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3382..3573)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4382..5464)
                     /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     5426..5445
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5465..5542)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5748..5767
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(5851..5856,1..14)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5851..5856,1..13)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gactcactat aggggaattg tgagcggata acaattcccc tctagaaata attttgttta
       61 actttaagaa ggagatatac catggcaact tcaactaaaa aattacataa agaacctgcg
      121 actttaatta aagcgattga tggtgatacg gttaaattaa tgtacaaagg tcaaccaatg
      181 acattcagac tattattggt tgatacacct gaaacaaagc atcctaaaaa aggtgtagag
      241 aaatatggtc ctgaagcaag tgcatttacg aaaaaaatgg tagaaaatgc aaagaaaatt
      301 gaagtcgagt ttgacaaagg tcaaagaact gataaatatg gacgtggctt agcgtatatt
      361 tatgctgatg gaaaaatggt aaacgaagca ttagttcgtc aaggcttggc taaagtttgc
      421 tatgtttaca aacctaacaa tacacatgaa caattattaa gaaaaagtga agcacaagcg
      481 aaaaaagaga aattaaatat ttggagcgaa gacaacgctg attcgggcca aactagtggt
      541 ggctcaggag ggggctccgg cggagggtct ggtggatcct tggggaatga aatcacgctg
      601 attatctttg gcgtgatggc cggagttatc ggtacaattc tgctgattag ctacggtatt
      661 cgtaacccaa gcgaattcaa gagctccgtc gacaagcttg cggccgcact cgagcaccat
      721 caccatcacc attgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
      781 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
      841 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg
      901 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
      961 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     1021 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     1081 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     1141 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     1201 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     1261 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     1321 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     1381 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact
     1441 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     1501 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     1561 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     1621 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     1681 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     1741 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     1801 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     1861 gcaggaacac tgccagcaca tcaacaatat tttcacctga atcaggatat tcttctaata
     1921 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     1981 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     2041 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     2101 catcgggctt cccatacaat cgatagattg tcgcacctga ttgtccgaca ttatcgcgag
     2161 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag
     2221 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     2281 gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     2341 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     2401 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     2461 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     2521 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     2581 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     2641 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     2701 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     2761 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     2821 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     2881 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     2941 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     3001 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     3061 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     3121 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     3181 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     3241 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     3301 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     3361 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     3421 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     3481 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     3541 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     3601 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     3661 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     3721 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     3781 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     3841 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     3901 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     3961 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     4021 cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa
     4081 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
     4141 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
     4201 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
     4261 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     4321 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     4381 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     4441 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     4501 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     4561 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     4621 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca
     4681 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     4741 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     4801 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     4861 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     4921 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     4981 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     5041 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     5101 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     5161 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     5221 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     5281 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     5341 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     5401 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     5461 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     5521 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     5581 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     5641 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
     5701 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
     5761 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     5821 tagaggatcg agatctcgat cccgcgaaat taatac
//
