LOCUS       sequence-243685-        6780 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To transform BL21-Gold(DE3) and be induced by IPTG for 
            His-proteinG-MNase fusion protein expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6780)
  TITLE     To transform BL21-Gold(DE3) and be induced by IPTG for 
            His-proteinG-MNase fusion protein expression.
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6780)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pET15bHis6-pG-MNase
FEATURES             Location/Qualifiers
     source          1..6780
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1..23
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             42..59
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             69..86
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             723..1169
                     /codon_start=1
                     /product="micrococcal nuclease from Staphylococcus aureus"
                     /label=MNase
                     /translation="ATSTKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLLVDTPETK
                     HPKKGVEKYGPEASAFTKKMVENAKKIEVEFDKGQRTDKYGRGLAYIYADGKMVNEALV
                     RQGLAKVAYVYKPNNTHEQLLRKSEAQAKKEKLNIWSEDNADSGQ"
     primer_bind     complement(1223..1241)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1237..1284
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1459..1464,1465..1481,1482..1487))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1514..1532)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1600..1704
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1705..1773,1774..2565)
                     /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(1923..1942)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2736..3324
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3225..3244
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3478..3495
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3510..3649
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3575..3594)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3735..3757
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3751..3942)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5254..6336)
                     /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     6298..6317
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6337..6414)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6620..6639
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6723..6742
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6723..6741
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6742..6766
                     /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)."
ORIGIN
        1 tttgtttaac tttaagaagg agatatacca tgggcagcag ccatcatcat catcatcaca
       61 gcagcggcct ggtgccgcgc ggcagccatt tgaagggtga aacgaccaca gaagcagtag
      121 acgcggccac tgccgagaag gtgtttaaac aatatgcgaa cgacaacgga gtcgatgggg
      181 aatggactta tgacgatgca accaaaactt tcactgttac ggagaagccc gaagtaattg
      241 acgccagcga acttaccccc gcggtaacga cgtataaact tgtaattaat ggtaagacgc
      301 tgaaggggga gaccaccact gaggccgtag atgcagctac agccgagaag gtctttaaac
      361 agtatgccaa tgataatggt gtcgacggtg agtggacgta cgatgatgca actaagacat
      421 tcaccgtaac agaaaagccg gaagttattg acgcgtcgga acttactcct gcggtgacca
      481 cttacaaact ggtaatcaac ggcaagacgc ttaagggaga aacaacgact aaagccgtcg
      541 acgccgaaac ggctgagaaa gctttcaagc aatacgccaa tgataatggg gtagacgggg
      601 tttggacgta tgatgacgcc accaagactt ttacagtcac ggaaatgggt ggtggaatga
      661 taacttcaca aacagataac ggcgtaaata gaagtggttc tgaagatcca acagtatata
      721 gtgcaacttc aactaaaaaa ttacataaag aacctgcgac attaattaaa gcgattgatg
      781 gtgatacggt taaattaatg tacaaaggtc aaccaatgac attcagacta ttattggttg
      841 atacacctga aacaaagcat cctaaaaaag gtgtagagaa atatggtcct gaagcaagtg
      901 catttacgaa aaaaatggta gaaaatgcaa agaaaattga agtcgagttt gacaaaggtc
      961 aaagaactga taaatatgga cgtggcttag cgtatattta tgctgatgga aaaatggtaa
     1021 acgaagcttt agttcgtcaa ggcttggcta aagttgctta tgtttataaa cctaacaata
     1081 cacatgaaca acttttaaga aaaagtgaag cacaagcaaa aaaagagaaa ttaaatattt
     1141 ggagcgaaga caacgctgat tcaggtcaat aaggatccgg ctgctaacaa agcccgaaag
     1201 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     1261 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggata tcccgcaaga
     1321 ggcccggcag taccggcata accaagccta tgcctacagc atccagggtg acggtgccga
     1381 ggatgacgat gagcgcattg ttagatttca tacacggtgc ctgactgcgt tagcaattta
     1441 actgtgataa actaccgcat taaagcttat cgatgataag ctgtcaaaca tgagaattct
     1501 tgaagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     1561 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     1621 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     1681 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     1741 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     1801 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     1861 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     1921 ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact cggtcgccgc
     1981 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     2041 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     2101 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     2161 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     2221 aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta
     2281 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     2341 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     2401 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     2461 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     2521 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     2581 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     2641 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     2701 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     2761 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     2821 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     2881 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     2941 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     3001 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     3061 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     3121 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     3181 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     3241 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     3301 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     3361 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     3421 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     3481 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     3541 tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     3601 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     3661 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     3721 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     3781 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     3841 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     3901 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     3961 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     4021 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     4081 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     4141 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     4201 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     4261 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     4321 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     4381 cgacaggagc acgatcatgc gcacccgtgg ccaggaccca acgctgcccg agatgcgccg
     4441 cgtgcggctg ctggagatgg cggacgcgat ggatatgttc tgccaagggt tggtttgcgc
     4501 attcacagtt ctccgcaaga attgattggc tccaattctt ggagtggtga atccgttagc
     4561 gaggtgccgc cggcttccat tcaggtcgag gtggcccggc tccatgcacc gcgacgcaac
     4621 gcggggaggc agacaaggta tagggcggcg cctacaatcc atgccaaccc gttccatgtg
     4681 ctcgccgagg cggcataaat cgccgtgacg atcagcggtc caatgatcga agttaggctg
     4741 gtaagagccg cgagcgatcc ttgaagctgt ccctgatggt cgtcatctac ctgcctggac
     4801 agcatggcct gcaacgcggg catcccgatg ccgccggaag cgagaagaat cataatgggg
     4861 aaggccatcc agcctcgcgt cgcgaacgcc agcaagacgt agcccagcgc gtcggccgcc
     4921 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
     4981 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
     5041 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
     5101 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
     5161 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     5221 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     5281 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5341 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     5401 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     5461 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     5521 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     5581 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     5641 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     5701 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     5761 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     5821 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     5881 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     5941 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     6001 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     6061 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     6121 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     6181 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     6241 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     6301 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     6361 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     6421 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     6481 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     6541 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     6601 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     6661 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa
     6721 attaatacga ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat
//
