LOCUS       sequence-415378-        7751 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NZ_CP025815 His14-TwinStrep-bdSUMO tagged DinG HNH protein (E. coli 
            codon optimized) in pET45b(+).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7751)
  AUTHORS   Altae-Tran H, Kannan S, Suberski AJ, Mears KS, Demircioglu FE, 
            Moeller L, Kocalar S, Oshiro R, Makarova KS, Macrae RK, Koonin EV, 
            Zhang F
  TITLE     Uncovering the functional diversity of rare CRISPR-Cas systems with 
            deep terascale clustering.
  JOURNAL   Science. 2023 Nov 24;382(6673):eadi1910. doi: 
            10.1126/science.adi1910. Epub 2023 Nov 23.
  PUBMED    37995242
REFERENCE   2  (bases 1 to 7751)
  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: "Science."; 
            date: "2023-11-24"; volume: "382(6673):eadi1910. doi"; pages: " 
            10.1126/science.adi1910. Epub 2023 Nov 23"
COMMENT     Sequence Label: pHS1184
FEATURES             Location/Qualifiers
     source          1..7751
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     207..226
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     310..329
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        310..328
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    329..353
                     /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             368..390
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             484..501
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(514..537,538..573,574..597)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     terminator      2575..2622
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2797..2802,2803..2819,2820..2825))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2852..2870)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2938..3042
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3043..3111,3112..3903)
                     /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(3261..3280)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4074..4662
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4563..4582
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4816..4833
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4848..4987
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4913..4932)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5073..5095
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5089..5280)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6592..7674)
                     /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     7636..7655
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(join(7675..7751,1))
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
       61 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
      121 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
      181 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
      241 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
      301 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
      361 aaataatttt gtttaacttt aagaaggaga tataccatgg gcagcagcca tcaccatcat
      421 tcaggccatc accataccgg acaccaccat cattcaggca gtcatcacca ttccagcagc
      481 ggcctggtgc cgcgcggcag ccatatggct agctggagcc atccgcagtt tgaaaaaggt
      541 ggtggtagcg gtggtggttc aggtggtagt gcatggtcac accctcagtt tgagaaagga
      601 tctgctgcgg gtggcgaaga agataagaaa ccggcaggtg gcgaaggtgg cggtgcccat
      661 atcaacctga aagtgaaagg tcaagacggc aacgaagtct ttttccgcat caaacgttct
      721 acccagctga aaaagctgat gaacgcatac tgtgaccgtc agtctgtaga catgaccgca
      781 attgctttcc tgtttgatgg tcgtcgcctg cgtgcggaac agaccccgga tgaactggag
      841 atggaagatg gcgacgaaat cgacgcaatg ctgcaccaga ctggtggctc aagtggatcc
      901 atgttcagat cgccagaaca agctgagttc aaccaatttg tagctaatca cataagtaat
      961 ccggatgcac cgctgttgat tgagggcgca actggcctgg gtaagacccg tgcctacctg
     1021 gcggcggtgt tccagaccga caaacgtgtg gcgatctgcc tggccacgaa cgcccttatc
     1081 gagcagattc tgaacagctc tgatctgccg tgggcgcagg agctggcgcc agataaaacc
     1141 gttgcggtgt tcagaagccg tcgttacttc gaagatgacc gtgaggcgta cgaagctcag
     1201 cgtgaggcgg cccaaatcgc cgatattctg atctgcaccg cgtcctccgt catcttcgac
     1261 caacgcttgt ccggtagcta taacggcgtt accaatcgcg acgttatcgt gttcgacgaa
     1321 gccgatcaga ttccgggtct ggcggctttg gcgagcgacc tgagcattga tcgtaaaacg
     1381 ttgcgcgact taaactgcgc ggcctcgacc gcacttgagg ttgcgggtaa gctgctgagc
     1441 ctgccgcagc tggatagcga aattcgcgca aaggctaaga tcatcgctga aattgcttcc
     1501 gaaccggaag tttggtataa acgtgtgggt atgaccgaag aaggtggcgt tagcgttatc
     1561 caccgtctgc ctggccgctt gctgaaaaaa attagtaatc gcccgagcac catttttatt
     1621 agcgcaacgt tgtcgatcaa cggccaattt aatgacttta agagggctat gggtattggc
     1681 gacagctctt cgttgtcacg tatcatagag ccgaaacacc acggtcatct gagctttagc
     1741 ttctttacgg acgatccggt tgattccgat gagtggctgg cgaccgtcgt ggagcaaatc
     1801 gaggcttcgg acaccccggt gctggtcgcc acaccgagcc ataagctggc aacggaatta
     1861 ggcgatagaa tttctggctc tacggtgcgt caacgtgatg aaacgatgac cgatgctgtt
     1921 ggtcgtatgg gcgacagaga cattgtgatc ggcgcaggtg catgggcggg tatggacacc
     1981 ccggtgcaat gggcgaccgt gatcatcccg cgtgttccat ttaccggtcc gaatgaactg
     2041 ttcgacacct ggaccgatga ggacgcgttt cgtattggcg acccgatgac cagctatttc
     2101 gatagcaaaa acgcggcgac ccgccgactc aagcaggttt tcggtcgtgg tttgcgccac
     2161 cccgacgctc gttgtgctat cgtcatttgc gatccgcgta tcagccagct gggcgacgtg
     2221 gccccagtgc gttttcgcga aggttggttc gagggtcgtc gcgtggaggt tgttcaaagc
     2281 aaagcggagc gcaacccggc actgcgccgt gacgcgctgc gtcatcatgg aaccgattgt
     2341 cagacctgtg gtcaccagcc gctgattctc cgcgaagttg aagtccacca ccttaatccg
     2401 attgctgagg cgaagggaat gatcgcgact tccatggacg acgtggcggt tctgtgccgt
     2461 atctgccatg cacgtgcgca caaagatggt aacaacgtaa ttccgctgga gcgtctgcgt
     2521 gagatcgcga aggcaaagtc tcgcacagaa gcccagcagc taattagttt ctgactagca
     2581 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
     2641 tccggatatc ccgcaagagg cccggcagta ccggcataac caagcctatg cctacagcat
     2701 ccagggtgac ggtgccgagg atgacgatga gcgcattgtt agatttcata cacggtgcct
     2761 gactgcgtta gcaatttaac tgtgataaac taccgcatta aagcttatcg atgataagct
     2821 gtcaaacatg agaattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt
     2881 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     2941 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     3001 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     3061 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     3121 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     3181 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     3241 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa
     3301 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     3361 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     3421 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     3481 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     3541 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca
     3601 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     3661 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     3721 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     3781 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     3841 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     3901 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     3961 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     4021 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     4081 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     4141 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     4201 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     4261 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     4321 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     4381 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     4441 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     4501 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     4561 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     4621 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     4681 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     4741 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     4801 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     4861 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     4921 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     4981 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     5041 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     5101 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     5161 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     5221 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     5281 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     5341 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     5401 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     5461 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     5521 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttacagac tttacgaaac
     5581 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     5641 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     5701 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtggcc aggacccaac
     5761 gctgcccgag atgcgccgcg tgcggctgct ggagatggcg gacgcgatgg atatgttctg
     5821 ccaagggttg gtttgcgcat tcacagttct ccgcaagaat tgattggctc caattcttgg
     5881 agtggtgaat ccgttagcga ggtgccgccg gcttccattc aggtcgaggt ggcccggctc
     5941 catgcaccgc gacgcaacgc ggggaggcag acaaggtata gggcggcgcc tacaatccat
     6001 gccaacccgt tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat cagcggtcca
     6061 atgatcgaag ttaggctggt aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg
     6121 tcatctacct gcctggacag catggcctgc aacgcgggca tcccgatgcc gccggaagcg
     6181 agaagaatca taatggggaa ggccatccag cctcgcgtcg cgaacgccag caagacgtag
     6241 cccagcgcgt cggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     6301 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     6361 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     6421 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     6481 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     6541 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     6601 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     6661 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     6721 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     6781 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     6841 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     6901 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     6961 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     7021 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     7081 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     7141 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     7201 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     7261 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     7321 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     7381 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     7441 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     7501 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     7561 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     7621 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     7681 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     7741 ttcgatggtg t
//
