LOCUS       sequence-380472-        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 poly-Leu-CP8B1 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-CP8B1
FEATURES             Location/Qualifiers
     source          1..5856
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     42..61
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        42..60
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    61..85
                     /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             100..122
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             132..578
                     /codon_start=1
                     /product="micrococcal nuclease from Staphylococcus aureus"
                     /label=MNase
                     /translation="ATSTKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLLVDTPETK
                     HPKKGVEKYGPEASAFTKKMVENAKKIEVEFDKGQRTDKYGRGLAYIYADGKMVNEALV
                     RQGLAKVCYVYKPNNTHEQLLRKSEAQAKKEKLNIWSEDNADSGQ"
     CDS             762..779
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(832..850)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      846..893
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      930..1385
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1017..1036)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1227..1248
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1477..2292)
                     /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     2200..2219
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2284..2303
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2414..3002
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2903..2922
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3156..3173
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3188..3327
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3253..3272)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3413..3435
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3429..3620)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4429..5511)
                     /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     5473..5492
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5512..5589)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5795..5814
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg
       61 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
      121 gatataccat ggcaacttca actaaaaaat tacataaaga acctgcgact ttaattaaag
      181 cgattgatgg tgatacggtt aaattaatgt acaaaggtca accaatgaca ttcagactat
      241 tattggttga tacacctgaa acaaagcatc ctaaaaaagg tgtagagaaa tatggtcctg
      301 aagcaagtgc atttacgaaa aaaatggtag aaaatgcaaa gaaaattgaa gtcgagtttg
      361 acaaaggtca aagaactgat aaatatggac gtggcttagc gtatatttat gctgatggaa
      421 aaatggtaaa cgaagcatta gttcgtcaag gcttggctaa agtttgctat gtttacaaac
      481 ctaacaatac acatgaacaa ttattaagaa aaagtgaagc acaagcgaaa aaagagaaat
      541 taaatatttg gagcgaagac aacgctgatt cgggccaaac tagtggtggc tcaggagggg
      601 gctccggcgg agggtctggt ggatccttgg ggaatgaaat cacgctgctc ttgatggtac
      661 tgctgggagc tctgcttggc gccctgctgg tagtactgat cggtattcgt aacccaagcg
      721 aattcaagag ctccgtcgac aagcttgcgg ccgcactcga gcaccatcac catcaccatt
      781 gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
      841 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
      901 gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
      961 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     1021 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     1081 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     1141 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     1201 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     1261 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     1321 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     1381 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     1441 atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa
     1501 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt
     1561 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg
     1621 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat
     1681 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag
     1741 tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc
     1801 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg
     1861 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc
     1921 cagcacatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt
     1981 tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt
     2041 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac
     2101 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc
     2161 atacaatcga tagattgtcg cacctgattg tccgacatta tcgcgagccc atttataccc
     2221 atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg
     2281 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca
     2341 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     2401 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     2461 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     2521 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     2581 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     2641 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     2701 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     2761 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     2821 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     2881 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     2941 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     3001 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     3061 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     3121 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     3181 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg
     3241 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     3301 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     3361 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     3421 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
     3481 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
     3541 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
     3601 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
     3661 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     3721 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     3781 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
     3841 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
     3901 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
     3961 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
     4021 ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc
     4081 acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg
     4141 ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg
     4201 ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc
     4261 ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc
     4321 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     4381 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     4441 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4501 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     4561 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     4621 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     4681 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
     4741 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     4801 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     4861 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     4921 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     4981 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     5041 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     5101 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     5161 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     5221 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     5281 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     5341 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     5401 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     5461 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     5521 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     5581 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     5641 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     5701 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     5761 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     5821 caaccgcacc tgtggcgccg gtgatgccgg ccacga
//
