LOCUS       sequence-415395-        9057 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  JAGBLH010000220 Cas8-HNH operon (E. coli codon optimized) with 
            His-tagged Cas8 in pET45b(+).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9057)
  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 9057)
  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: pHS1928
FEATURES             Location/Qualifiers
     source          1..9057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(join(152..157,158..174,175..180))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(207..225)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        293..397
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(398..466,467..1258)
                     /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(616..635)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1429..2017
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1918..1937
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2171..2188
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2203..2342
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2268..2287)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2428..2450
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2444..2635)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3947..5029)
                     /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     4991..5010
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5030..5107)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5313..5332
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5416..5435
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5416..5434
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5435..5459
                     /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             5474..5496
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6196..6210
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             7161..7181
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     terminator      8987..9034
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 atatcccgca agaggcccgg cagtaccggc ataaccaagc ctatgcctac agcatccagg
       61 gtgacggtgc cgaggatgac gatgagcgca ttgttagatt tcatacacgg tgcctgactg
      121 cgttagcaat ttaactgtga taaactaccg cattaaagct tatcgatgat aagctgtcaa
      181 acatgagaat tcttgaagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
      241 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
      301 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
      361 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
      421 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
      481 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
      541 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
      601 cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca
      661 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
      721 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
      781 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
      841 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
      901 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt
      961 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     1021 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     1081 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     1141 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     1201 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     1261 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     1321 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     1381 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     1441 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     1501 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     1561 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     1621 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     1681 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     1741 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     1801 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     1861 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     1921 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     1981 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     2041 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     2101 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     2161 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     2221 ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct
     2281 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     2341 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     2401 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     2461 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     2521 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     2581 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     2641 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     2701 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     2761 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     2821 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     2881 tccggaacat aatggtgcag ggcgctgact tccgcgttta cagactttac gaaacacgga
     2941 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     3001 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     3061 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggccaggac ccaacgctgc
     3121 ccgagatgcg ccgcgtgcgg ctgctggaga tggcggacgc gatggatatg ttctgccaag
     3181 ggttggtttg cgcattcaca gttctccgca agaattgatt ggctccaatt cttggagtgg
     3241 tgaatccgtt agcgaggtgc cgccggcttc cattcaggtc gaggtggccc ggctccatgc
     3301 accgcgacgc aacgcgggga ggcagacaag gtatagggcg gcgcctacaa tccatgccaa
     3361 cccgttccat gtgctcgccg aggcggcata aatcgccgtg acgatcagcg gtccaatgat
     3421 cgaagttagg ctggtaagag ccgcgagcga tccttgaagc tgtccctgat ggtcgtcatc
     3481 tacctgcctg gacagcatgg cctgcaacgc gggcatcccg atgccgccgg aagcgagaag
     3541 aatcataatg gggaaggcca tccagcctcg cgtcgcgaac gccagcaaga cgtagcccag
     3601 cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     3661 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     3721 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     3781 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     3841 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     3901 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     3961 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4021 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     4081 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     4141 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     4201 cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa
     4261 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     4321 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     4381 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     4441 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     4501 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     4561 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     4621 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     4681 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     4741 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     4801 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     4861 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     4921 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     4981 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     5041 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     5101 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     5161 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     5221 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     5281 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     5341 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc
     5401 tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc
     5461 ctctagaaat aattttgttt aactttaaga aggagatata ccatggcagc taataaaaag
     5521 gcaaccaatg tcacgctaaa atcccgcccc gaaaatctat catttgctag gtgtttaaac
     5581 acgactgagg cgaaattctg gcagaccgat tttctgaaac gccatacgtt caaattaccg
     5641 ctgctgatta ccgataaggc ggtcctcgcg agcaaaggcc acgaaatgcc gcctgataag
     5701 ctggaaaaag agatcatgga cccgaatccg caaaagtccc agtcttgtac cctgtcgacc
     5761 gagtgcgata cgctgcgtat tgactttggt attaaagtgc tgccggttaa agaaagcatg
     5821 tacagctgca gcgattacaa ctatcgtacc gcaatttatc agaaaatcga cgaatacatc
     5881 gcggaggacg gcttcttgac cttggcgaag cgctatgtga ataacatcgc gaatgcccgt
     5941 tttttgtggc gtaatcgtaa aggtgctgag atcatcgaaa ccattgttac cattgaggac
     6001 aaggagtacc cgagctttaa tagcaagtcc ttcaacctgg atactttcgt ggaggacaac
     6061 gccaccatca acgaaatcgc tcagcagatt gccgatacgt ttgctggcaa gcgcgaatac
     6121 ttgaatattt acgtcacctg ctttgttaag atcggttgtg caatggaagt gtatccatcg
     6181 caagaaatga cctttgatga cgatgacaag ggtaagaaac tgttcaagtt cgagggtagc
     6241 gcaggtatgc atagccaaaa gatcaacaac gcgctgcgta ccattgacac atggtatccg
     6301 gactacacca cctatgaatt tccgattccg gtagagaact acggcgcggc gcgttcaatc
     6361 ggcatcccgt tcagaccaga caccaaatct ttctataaac ttatcgaccg catgattctg
     6421 aaaaacgagg atctgccgat cgaggacaag cactacgtta tggcaatttt gattcgaggc
     6481 ggcatgttca gcaagaaaca agaaaaataa gcattcagga ggaaacgata tgtttagtca
     6541 aattcttatc ataaaaccag gcacaggcat atcccccaat attataatct cagaagatat
     6601 atttccagtt ctgcatagct tgttcgtgga gcacgataaa aagtttggta tcacgttccc
     6661 ggcatactcc tttgataaaa agggccatct gggtaatatc attgaagttc tgagcgaaga
     6721 taaagaggcg ctggcgagcc tgtgtctgga ggagcacttg gcggaagtca ccgactatgt
     6781 gaaagttaag aaggagatca cgttcaccga cgactacgtg ttgtttaaac gcattagaga
     6841 agagaaccaa tatgaaacca ctgcccgtcg tatgcgtaaa cgcggtcaca ccgaactcgg
     6901 ccgtccgctg gagatgcata ttaagaagaa gaaccagcaa attttctgcc acgcgtacat
     6961 caaggtgaaa agcgcatcca ccggtcagag ctataacatc tttcttgctc cgaccgacat
     7021 caaacacggc tcgttttccg cttatggact tcttcgaggt gatactcatg cttagaagca
     7081 agcatcacca tcattcaggc catcaccata ccggacacca ccatcattca ggcagtcatc
     7141 accataccgg gggctcaggc gagaacctgt attttcaggg ttctggtgga cttagaaata
     7201 aaatattggc tgccatatcg caaaaaatcc ctgaggaaca aaaaataaat aagtatattg
     7261 aagggctgtt tcaaagcatt gataagaacc accttgctac gcatgtggcc aaattcaccg
     7321 aaaccaacag cccgggtaat ataggtgcgt atgatatctt gtccagcgac atgaactgcg
     7381 gttacctgga caccgcaaat gccggttgga aagaaccgga tatcgttacc aacgatgcga
     7441 aatacaaacg tcctcagggc tttgttgcta tggaaatgtc cgacggccgt accgtgatgg
     7501 agcacctaca agaagattct gcagaattaa gacacgagat ggaagagttg accgataagt
     7561 acgacgagat ccgtgacggt attttgaaca tgccgagcat gcagccgtat cgcacgaacc
     7621 agtttatcaa gcaagttttc ttcccggtcg gtggtagcta tcatctcctc agcattctcc
     7681 caagcaccgt actgaattac gaggtgagcg accgcctgta tcgtagcaag atcccgaaga
     7741 ttcgcctgcg cttgctgtcg agcaacgcgg catcgacgac cggtagccgt ctggtttcca
     7801 agaacaagtg gccgcttgtt ttccaagcgc tgccgccaaa gttcctggaa aaaaacctgg
     7861 ctaaagcgct ggacaaggag tatctgctgc cggacatcaa catcgacgag ttggaaggcg
     7921 ttgataatgg ttgtctgatt gatgaggccc tgttaccgct gatcatcgat gagggcaaac
     7981 gcaagggtga aggcaattat cgtccgcgtc acctgcgtga cgaacgtaaa gaggagactg
     8041 tccaggcgtt tttggacaaa tacggctact gcaatattcc ggtgggctac gaggtgcatc
     8101 atattgtgcc gctgtctcag ggcggtgcgg atagtatcaa aaacatgatt atgctgtcta
     8161 tcgaacacca cgaaagggtt actgaagctc atgcatcata tttcaagtgg agaaatacat
     8221 gatgaaggga tatatactgc ttgaaaaagt aaatattgaa aacgctaatg catttaataa
     8281 cattatagta ggaatcccgg ctatcacctc attcctgggc tttgcaagag cgctagagcg
     8341 caaactgaac gcgaaagaga tcgctatccg tatcaacggt gttggcctgg aatttcatga
     8401 atatgaatta aaaggttaca agaacaaacg tggtcagtat gtgaccagct gtccgctgcc
     8461 gggttccatt ccaggtcaga acgagaagaa gctggacgct cacatcatga atcaggcata
     8521 tattgatctg aatatgagct ttttgctgga ggtggagggt ccgcatgttg atatgtctac
     8581 gtgcaaatct attaaatcga ccatggaaac gctgcgtatt gcgggtggca ttattcgcaa
     8641 ctacaagaag atccgcttga ttgacaccct tgccgatatc ccgtacggct acttcttgac
     8701 cctccgtcag gataatctga atgatgcggc aggcgacgac atgctggata agatgattca
     8761 tgcgttgcaa caagaggaca ctttggttcc gattgccgtt ggcttcaagg cgctgagcga
     8821 agtcggtcac gtggagggcc aacgtgatcc ggaaaaggac cactgcttcg tggagtccat
     8881 cttcagcctg ggcggttttg aatgtagcaa aatcctggag gacatcaaca gctgcctgtg
     8941 gcgctataaa acagaagaag ggctatatct ttgcaccata atttgactag cataacccct
     9001 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccgg
//
