LOCUS       sequence-380471-        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-G83I 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-G83I
FEATURES             Location/Qualifiers
     source          1..5856
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     12..31
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        12..30
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    31..55
                     /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             70..92
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             102..548
                     /codon_start=1
                     /product="micrococcal nuclease from Staphylococcus aureus"
                     /label=MNase
                     /translation="ATSTKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLLVDTPETK
                     HPKKGVEKYGPEASAFTKKMVENAKKIEVEFDKGQRTDKYGRGLAYIYADGKMVNEALV
                     RQGLAKVCYVYKPNNTHEQLLRKSEAQAKKEKLNIWSEDNADSGQ"
     CDS             732..749
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(802..820)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      816..863
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      900..1355
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(987..1006)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1197..1218
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1447..2262)
                     /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     2170..2189
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2254..2273
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2384..2972
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2873..2892
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3126..3143
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3158..3297
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3223..3242)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3383..3405
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3399..3590)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4399..5481)
                     /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     5443..5462
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5482..5559)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5765..5784
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct
       61 agaaataatt ttgtttaact ttaagaagga gatataccat ggcaacttca actaaaaaat
      121 tacataaaga acctgcgact ttaattaaag cgattgatgg tgatacggtt aaattaatgt
      181 acaaaggtca accaatgaca ttcagactat tattggttga tacacctgaa acaaagcatc
      241 ctaaaaaagg tgtagagaaa tatggtcctg aagcaagtgc atttacgaaa aaaatggtag
      301 aaaatgcaaa gaaaattgaa gtcgagtttg acaaaggtca aagaactgat aaatatggac
      361 gtggcttagc gtatatttat gctgatggaa aaatggtaaa cgaagcatta gttcgtcaag
      421 gcttggctaa agtttgctat gtttacaaac ctaacaatac acatgaacaa ttattaagaa
      481 aaagtgaagc acaagcgaaa aaagagaaat taaatatttg gagcgaagac aacgctgatt
      541 cgggccaaac tagtggtggc tcaggagggg gctccggcgg agggtctggt ggatccttgg
      601 ggaatgaaat cacgctgatt atctttggcg tgatggccat agttatcggt acaattctgc
      661 tgattagcta cggtattcgt aacccaagcg aattcaagag ctccgtcgac aagcttgcgg
      721 ccgcactcga gcaccatcac catcaccatt gagatccggc tgctaacaaa gcccgaaagg
      781 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
      841 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga
      901 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
      961 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     1021 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     1081 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     1141 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     1201 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     1261 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     1321 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
     1381 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat
     1441 taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     1501 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     1561 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     1621 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     1681 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac
     1741 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     1801 tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
     1861 aatcgaatgc aaccggcgca ggaacactgc cagcacatca acaatatttt cacctgaatc
     1921 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
     1981 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     2041 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     2101 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     2161 tccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     2221 tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact
     2281 gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt
     2341 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2401 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     2461 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     2521 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     2581 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     2641 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     2701 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     2761 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     2821 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     2881 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     2941 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3001 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     3061 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     3121 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     3181 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     3241 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     3301 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     3361 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     3421 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     3481 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     3541 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     3601 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     3661 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     3721 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     3781 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     3841 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     3901 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     3961 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     4021 gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg
     4081 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     4141 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     4201 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     4261 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     4321 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     4381 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4441 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     4501 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     4561 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     4621 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     4681 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     4741 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     4801 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     4861 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     4921 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     4981 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     5041 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     5101 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     5161 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     5221 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     5281 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     5341 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     5401 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     5461 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     5521 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     5581 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     5641 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     5701 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     5761 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     5821 ccacgatgcg tccggcgtag aggatcgaga tctcga
//
