LOCUS       sequence-326091-        7778 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a thermostable Cas9 enzyme and gRNA in Thermus 
            thermophilus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7778)
  AUTHORS   Adalsteinsson BT, Kristjansdottir T, Merre W, Helleux A, Dusaucy J, 
            Tourigny M, Fridjonsson O, Hreggvidsson GO
  TITLE     Efficient genome editing of an extreme thermophile, Thermus 
            thermophilus, using a thermostable Cas9 variant.
  JOURNAL   Sci Rep. 2021 May 5;11(1):9586. doi: 10.1038/s41598-021-89029-2.
  PUBMED    33953310
REFERENCE   2  (bases 1 to 7778)
  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: "Sci Rep."; 
            date: "2021-05-5"; volume: "11(1):9586. doi"; pages: " 
            10.1038/s41598-021-89029-2"
COMMENT     Sequence Label: pTTCC
FEATURES             Location/Qualifiers
     source          1..7778
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(3485..3502)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3655..3726
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     RBS             3912..3920
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     promoter        join(6564..6569,6570..6586,6587..6593)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     RBS             6608..6616
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             6622..6795
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGE"
     primer_bind     complement(6642..6659)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6642..6658)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6651..6673)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(7147..7735)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7227..7246)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acgaaaactc acgttaaggg attttggtca tgaattcgct agagtcgacc ccggggcgcg
       61 aggggaggag ggctcgaggc ccccctcttg caagcgcttt caacagatgc tatactcccg
      121 ggcaaagatg accaaggctc gccccaccat cgccgaaact agttcttgga ggagaaacgc
      181 atatgcgcta caagatcggc ctggacatcg gcatcaccag cgtgggctgg gcggtgatca
      241 acctcgatat tccgcggatc gaggacctgg gtgtccggat tttcgatcgc gccgaaaatc
      301 cgcaaacggg agaaagcctg gccctgcccc gtcggctggc gcgcagcgcc cggcgtcgcc
      361 tccgccgccg gaaacatcgc ctggaacgca ttcgccgtct gatcatccgg gaagggatcc
      421 tgacgaagga ggagctggac aagctgtttg aggagaagca cgagatcgat gtctggcagc
      481 tccgggtgga ggcgctggac cggaagctca acaacgacga gctggcgcgg gtcctgctgc
      541 atctggccaa gcgccggggc ttcaaaagca accgcaagtc ggaacgctcg aacaaggaga
      601 actcgacgat gctgaagcac atcgaggaga accgcgccat cctgtcctcg tatcgcaccg
      661 tcggcgagat gatcgtgaaa gacccgaagt ttgccctgca caaacgtaac aagggcgaga
      721 actacaccaa taccattgcc cgcgacgacc tggagcggga gatccggctc atcttctcga
      781 agcagcgcga atttgggaac atgtcgtgca ccgaggagtt cgagaacgaa tacattgcca
      841 tctgggcctc gcagcggccg gtggcgtcga aggacgacat tgagaagaag gtggggttct
      901 gcacgttcga accgaaggag aagcgggcgc cgaaagccac gtacacgttc cagtcgttca
      961 tcgcgtggga gcacatcaac aaactccggc tgatctcgcc gtcggggacc cgcggcctga
     1021 cggatgagga acggcgcctc ctgtacgagc aggccttcca gaagaacaag atcacgtacc
     1081 acgacatccg cacgctgctg cacctgccgg atgatacgta ttttaagggc attgtgtatg
     1141 accgtggcga gtcgcggaaa cagaacgaga acatccggtt cctggagctc gacgcgtacc
     1201 accaaatccg gaaggccgtg gacaaagtgt acggcaaagg caaatcgtcc tccttcctgc
     1261 cgatcgactt cgataccttc ggatatgccc tgaccctgtt caaagacgac gcggacattc
     1321 ggagctatct ccggaacgaa tacgagcaga acggtaaacg catgccgaac ctggccaaca
     1381 aggtctacga caatgaactc atcgaagaac tgctcaacct gtccttcacg aagtttggcc
     1441 acctgagcct gaaggccctg cgcagcatcc tcccctacat ggagcagggc gaagtgtaca
     1501 gctcggcctg tgagcgcgcg ggatacacct tcacgggccc caagaagaag caaaagacga
     1561 tgctgctgcc gaacattccg cccatcgcca accccgtggt catgcgggcc ctgacgcagg
     1621 cccggaaggt ggtgaacgcg atcatcaaga agtacggaag ccccgtgtcg atccacatcg
     1681 aactggcccg ggacctgtcg cagacgttcg acgaacgccg caaaaccaag aaggaacagg
     1741 atgagaaccg caagaagaac gaaacggcca tccgccagct gatggagtac ggactgacgc
     1801 tgaatccgac gggtcacgac atcgtgaaat tcaaactgtg gtcggagcag aacggccgct
     1861 gcgcctattc gctgcagccc atcgaaatcg agcggctcct ggaaccgggc tacacggagg
     1921 tggaccacgt cattccgtac tcgcgctccc tggatgactc ctacaccaac aaggtgctgg
     1981 tcctcacgaa ggagaatcgc gagaagggta accgcatccc ggccgagtat ctgggcgtgg
     2041 gcacggagcg ctggcagcag ttcgagacgt ttgtcctgac gaacaaacag ttcagcaaga
     2101 agaaacgcga ccggctgctg cgcctgcatt acgacgaaaa cgaggaaacg gagttcaaga
     2161 accgcaacct gaacgacacg cgctacatca gccggttctt cgcgaacttc atccgcgagc
     2221 acctcaagtt cgcggaatcg gacgataagc agaaggtgta caccgtcaac ggccgtgtga
     2281 cggcgcatct gcgctcgcgc tgggagttca acaagaaccg cgaagaatcg gatctgcacc
     2341 acgccgtgga cgccgtgatc gtggcctgca cgaccccgtc ggacatcgcc aaggtgaccg
     2401 ccttctatca gcgccgcgag cagaacaagg aactggccaa aaagaccgaa ccgcatttcc
     2461 cgcagccgtg gccgcatttt gccgatgagc tgcgcgcgcg cctgtcgaag cacccgaaag
     2521 aatcgatcaa ggccctgaac ctggggaact atgacgacca gaagctggag tccctgcagc
     2581 ccgtgttcgt ctcccggatg cccaagcgca gcgtcacggg cgcggcccat caggaaacgc
     2641 tgcgccgcta cgtgggcatc gacgaacgtt cggggaagat tcagacggtg gtgaaaacca
     2701 agctgagcga aatcaagctc gatgcgtcgg gccactttcc catgtacggg aaggagagcg
     2761 atccccgtac ctacgaggcg atccggcagc gcctgctgga acacaacaac gaccccaaga
     2821 aagcctttca ggagcccctc tacaagccga agaagaacgg tgagccgggc ccggtcattc
     2881 ggaccgtgaa gatcatcgac acgaagaacc aggtgattcc gctgaacgat gggaaaacgg
     2941 tggcctacaa ctcgaatatc gtgcgggtgg acgtgttcga gaaggacggc aagtactact
     3001 gcgtccccgt ctatacgatg gacatcatga agggcatcct gcccaataag gccattgagc
     3061 cgaacaagcc gtacagcgaa tggaaggaga tgacggagga ctacacgttc cggttctcgc
     3121 tctaccccaa cgatctgatc cgcatcgaac tgccgcgtga aaagatcatt aagacggccg
     3181 gcggcgagga aatcaaaatc aaggatctct ttgcgtacta caaaaccatc cactcgggga
     3241 ccgccggcct ggagctggtc agccacgact gctcgttcag cctgtccggc gtgggttcgc
     3301 gcacgctgaa gcgctttgag aagtaccagg tggacgtgct gggcaacatc tacaaagtgc
     3361 gcggcgagaa gcgggtgggc ctggcgagct cggcccacag caagaccggt gagacgattc
     3421 gcccgctgca gtcgacgcgg gactagaagc ttggctgttt tggcggatga gagaagattt
     3481 tcagcctgat acagattaaa tcagaacgca gaagcggtct gataaaacag aatttgcctg
     3541 gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta
     3601 gcgccgatgg tagtgtgggg tctccccatg cgagagtagg gaactgccag gcatcaaata
     3661 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
     3721 gctctcctgc tagcgggagt ataacagaaa ccttaaggcc cgaccgcttg acaagggcgc
     3781 gtgaggtttt tacgatagcg ccggatgcgg ggaaaaaggg ctccttttgg ggggttttcc
     3841 ccgcaccggg cggacctggg cggagaggaa acgcggcaac tcgcccgtct cgggttcccg
     3901 cccacgaccc ttaaggaggt gtgaggatta tgaatggacc aataataatg actagagaag
     3961 aaagaatgaa gattgttcat gaaattaagg aacgaatatt ggataaatat ggggatgatg
     4021 ttaaggctat tggtgtttat ggctctcttg gtcgtcagac tgatgggccc tattcggata
     4081 ttgagatgat gtgtgtcatg tcaacagagg aagcagagtt cagccatgaa tggacaaccg
     4141 gtgagtggaa ggtggaagtg aatttttata gcgaagagat tctactagat tatgcatctc
     4201 aggtggaatc agattggccg cttacacatg gtcaattttt ctctattttg ccgatttatg
     4261 attcaggtgg atacttagag aaagtgtatc aaactgctaa atcggtagaa gcccaaaagt
     4321 tccacgatgc gatttgtgcc cttatcgtag aagagctgtt tgaatatgca ggcaaatggc
     4381 gtaatattcg tgtgcaagga ccgacaacat ttctaccatc cttgacggta caggtagcaa
     4441 tggcaggtgc catgttgatt ggtctgcatc atcgcatctg ttatacgacg agcgcttcgg
     4501 tcttaactga agcagttaag caatcagatc ttccttcagg ttatgaccat ctgtgccagt
     4561 tcgtaatgtc tggtcaactt tccgactctg agaaacttct ggaatcgcta gagaatttct
     4621 ggaatgggat tcaggagtgg acagaacgac acggatatat agtggatgtg tcaaaacgca
     4681 taccattttg acaattgctg cagactagat gggggttgcc agcgcctcca atttttcaag
     4741 tgaaatctat aacgcctgtt ggcatgcctc agttgacccc attgaccctt ctctctggag
     4801 gtggtagcta gaggggaatg gtcccaccct ggagccccac cccttgccaa ccctgggccc
     4861 ggtataatgc cgggcaatgg gacgaccggg ccccctgggg gcccaaggcg gcagggccgc
     4921 gtgaggacca aaaagagagg ctcccgctgg gagggaaccc aaattccagc aaccttttcc
     4981 ttcgcctaag gttatcccct cccagcggga aaggcaatag gcgccaaggc ggcgcgcggg
     5041 ggagtgccgt ctccgacgcg cgaaacggga ggggtttatg ccccaagaag gagttgaagg
     5101 gaagagctat aaagaacagc cggaaggact caccgagcgg ggccgggcct tcctcgaggc
     5161 cctggcccgg aggaagcggg cgaggggcga ggagcttccg cccctctacc tgaagctcct
     5221 ggagggggcc gcgcccccgg agaagggggt ccatgagccc ccgcccgagg aggcgcctcc
     5281 cccggaggac ctccgccgga agctccggga ggcgccgcct tccccggccc tccccaaccg
     5341 acaggagctc tccgcctccc cgcccccgcc cgagatgagg cgggacgcct ggagcctcgc
     5401 cgaccgcctc ctggaggagg gggagcggcg gggcaccctg ccagggcttt ccgagcggga
     5461 gcggagggtg taccggaccc tcctcgccct gggcctcgag gtcctggccc ggaggctggg
     5521 cccggggagg cccttgccca ggaacctgtc ccaggtctcc ttcttcgccg tgaacgacgc
     5581 cctggccgtg gccctgggaa tccccccggc ctccctctac cgggtcctgg cctccctgga
     5641 ggccaagggg ctcatccgcc ggagggcctg gcgcaccccg gccaccctca agggccggac
     5701 gggggtctac gccggcggga ccctctacgc cgtgcgcctg ccccaccggg aggcccgccc
     5761 ccgcctggac ccggaggact tccgccaccc ctggcgggac ctggaggggg acgcccgtca
     5821 ggggcgcacc gcctggagct tgagagagtc atatacaagt cctcctaagg aggactccgg
     5881 ggtcctccag ctcctccttc ggttttcgtt atcccctggc gaagccgaaa ctccgttagc
     5941 tttagactct ctcaccgccc tcctccgggc ccgccccgcc gagcgccggg ccctggtgga
     6001 ggccctcgcc ctctccctgg cccgggagtt ccgggacccc gggagcgtgc gcttctacgc
     6061 ctgggtcctc tggaacgccc tccgggccga gctctacggc ctgatggagg gggccctcga
     6121 ggccgtcgcc tgggcggtcc ggcgggcgcg ggaggccgcg gccaaggtcc tctggagccc
     6181 gaggggcgag ggggtccggc gccccggggc cctcctcgcc cacctcctcc gggagcgggg
     6241 cctcctggag ctcttccgcc aggcccctca gtggcgggtg gcgtagggct cccgccgggc
     6301 taagctggag gtagcccatc cagggcctct ggggggtggc ttgtgtatca agactggctg
     6361 ttcaaggact gcccaggggg ctggcgcctc cgctaccgcc ggaacggggg cggctacgag
     6421 gcctcccggg acggtgagag gtgggagaag gtcccctcgg tcacggaggt tacggggcgc
     6481 ctgaacaaga accttcagga ctgggccgtg cgccaggtgg tggagtacct ccagggggag
     6541 ctggtcccgg gggtactgag ctgttgacaa ttaatcatcc ggctcgtata atgtgtggaa
     6601 cgtcttctaa ggaggtcaca tatgaccatg attacggatt cactggccgt cgttttacaa
     6661 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     6721 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     6781 agcctgaatg gcgaatagga agactagtca tagttcccct gagattatcg ctgtggtata
     6841 atatcatacc acagcaatga tctcagggtt actatgataa gggctttctg cctaaggcag
     6901 actgacccgc ggcgttgggg atcgcctgtc gcccgctttt ggcgggcatt ccccatcctt
     6961 gcgcaaactc agaccttggc ggaaaacgct aaggtctttt ggtaccaccg aattccgagc
     7021 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     7081 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     7141 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     7201 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     7261 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     7321 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     7381 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     7441 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     7501 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     7561 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     7621 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     7681 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     7741 tcctttgatc ttttctacgg ggtctgacgc tcagtgga
//
