LOCUS       sequence-270279-        5425 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Micrococcal nuclease.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5425)
  AUTHORS   Yan Q, Shields EJ, Bonasio R, Sarma K
  TITLE     Mapping Native R-Loops Genome-wide Using a Targeted Nuclease 
            Approach.
  JOURNAL   Cell Rep. 2019 Oct 29;29(5):1369-1380.e5. doi: 
            10.1016/j.celrep.2019.09.052.
  PUBMED    31665646
REFERENCE   2  (bases 1 to 5425)
  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.celrep.2019.09"; journalName: "Cell Rep"; date: 
            "2019-10-29- 29"; volume: "29"; issue: "5"; pages: "1369-1380.e5"
COMMENT     Sequence Label: GST-MNase
FEATURES             Location/Qualifiers
     source          1..5425
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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             76..729
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     687..709
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             736..759
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             778..1224
                     /codon_start=1
                     /product="micrococcal nuclease from Staphylococcus aureus"
                     /label=MNase
                     /translation="ATSTKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLLVDTPETK
                     HPKKGVEKYGPEASAFTKKMVENAKKIEVEFDKGQRTDKYGRGLAYIYADGKMVNEALV
                     RQGLAKVAYVYKPNNTHEQHLRKSEAQAKKEKLNIWSEDNADSGQ"
     primer_bind     complement(1293..1315)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1460..1478)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1546..1650
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1651..1719,1720..2511)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1869..1888)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2682..3270
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3171..3190
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3424..3441
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3514..3591
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3592..4674
                     /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     complement(3611..3630)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4723..4728,4729..4746,4747..4753)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4761..4777
                     /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)."
     primer_bind     4785..4801
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4785..4801
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4797..5324
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4817..4834)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4817..4833)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4826..4848)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag tattcatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
      121 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
      181 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
      241 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
      301 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
      361 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
      421 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
      481 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
      541 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
      601 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
      661 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
      721 cctccaaaat cggatctgga agttctgttc caggggcccc tgggatcccc ggaattcgca
      781 acttcaacta aaaaattaca taaagaacct gcgactttaa ttaaagcgat tgatggtgat
      841 acggttaaat taatgtacaa aggtcaacca atgacattca gactattatt ggttgataca
      901 cctgaaacaa agcatcctaa aaaaggtgta gagaaatatg gtcctgaagc aagtgcattt
      961 acgaaaaaaa tggtagaaaa tgcaaagaaa attgaagtcg agtttgacaa aggtcaaaga
     1021 actgataaat atggacgtgg cttagcgtat atttatgctg atggaaaaat ggtaaacgaa
     1081 gctttagttc gtcaaggctt ggctaaagtt gcttatgttt acaaacctaa caatacacat
     1141 gaacaacatt taagaaaaag tgaagcacaa gcgaaaaaag agaaattaaa tatttggagc
     1201 gaagacaacg ctgattcagg tcaataactc gagcggccgc atcgtgactg actgacgatc
     1261 tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     1321 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     1381 ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat
     1441 aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     1501 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     1561 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     1621 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     1681 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     1741 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     1801 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     1861 aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt
     1921 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     1981 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     2041 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     2101 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     2161 ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa
     2221 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     2281 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     2341 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     2401 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     2461 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     2521 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     2581 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     2641 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     2701 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     2761 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     2821 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     2881 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     2941 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     3001 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     3061 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     3121 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     3181 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     3241 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     3301 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     3361 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     3421 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
     3481 catctgtgcg gtatttcaca ccgcataaat tccgacacca tcgaatggcg caaaaccttt
     3541 cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca
     3601 gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg
     3661 gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg
     3721 gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg
     3781 attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt
     3841 aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc
     3901 gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc
     3961 attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt
     4021 ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat
     4081 gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg
     4141 ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa
     4201 tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg
     4261 tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg
     4321 gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc
     4381 gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc
     4441 ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc
     4501 ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg
     4561 gtgaaaagaa aaaccacgct agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     4621 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     4681 cgcaattaat gtaagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
     4741 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
     4801 ccatgattac ggattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
     4861 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
     4921 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgctttg
     4981 cctggtttcc ggcaccagaa gcggtgccgg aaagctggct ggagtgcgat cttcctgagg
     5041 ccgatactgt cgtcgtcccc tcaaactggc agatgcacgg ttacgatgcg cccatctaca
     5101 ccaacgtgac ctatcccatt acggtcaatc cgccgtttgt tcccacggag aatccgacgg
     5161 gttgttactc gctcacattt aatgttgatg aaagctggct acaggaaggc cagacgcgaa
     5221 ttatttttga tggcgttgga attagcttat cgactgcacg gtgcaccaat gcttctggcg
     5281 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg
     5341 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt
     5401 ctggcaaata ttctgaaatg agctg
//
