LOCUS       sequence-366871-        8896 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Thiomicrospira Cas6-RT-Cas1 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8896)
  AUTHORS   Wang JY, Hoel CM, Al-Shayeb B, Banfield JF, Brohawn SG, Doudna JA
  TITLE     Structural coordination between active sites of a CRISPR reverse 
            transcriptase-integrase complex.
  JOURNAL   Nat Commun. 2021 May 6;12(1):2571. doi: 10.1038/s41467-021-22900-y.
  PUBMED    33958590
REFERENCE   2  (bases 1 to 8896)
  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: "Nat 
            Commun."; date: "2021-05-6"; volume: "12(1):2571. doi"; pages: " 
            10.1038/s41467-021-22900-y"
COMMENT     Sequence Label: pET_His6-MBP-TEV-Cas6-RT-Cas1_WT
FEATURES             Location/Qualifiers
     source          1..8896
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(18..36)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        104..208
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(209..277,278..1069)
                     /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(427..446)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1240..1828
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1729..1748
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1982..1999
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2014..2153
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2079..2098)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2239..2261
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2255..2446)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     3904..3923
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4007..4026
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4007..4025
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             4114..4136
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4156..4173
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4183..5283
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     5257..5280
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             5338..5358
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(8432..8450)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      8446..8493
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(8859..8864,8865..8881,8882..8887))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
ORIGIN
        1 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
       61 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      121 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
      181 gcttcaataa cattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      241 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
      301 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
      361 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
      421 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg
      481 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
      541 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
      601 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
      661 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
      721 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
      781 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
      841 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
      901 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
      961 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1021 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1081 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1141 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     1201 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1261 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1321 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     1381 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     1441 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     1501 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     1561 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     1621 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     1681 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     1741 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     1801 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     1861 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     1921 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     1981 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     2041 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     2101 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     2161 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     2221 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     2281 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     2341 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     2401 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     2461 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     2521 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     2581 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     2641 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     2701 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     2761 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     2821 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     2881 tcaacgacag gagcacgatc atgcgcaccc gtggccagga cccaacgctg cccgagatgc
     2941 gccgcgtgcg gctgctggag atggcggacg cgatggatat gttctgccaa gggttggttt
     3001 gcgcattcac agttctccgc aagaattgat tggctccaat tcttggagtg gtgaatccgt
     3061 tagcgaggtg ccgccggctt ccattcaggt cgaggtggcc cggctccatg caccgcgacg
     3121 caacgcgggg aggcagacaa ggtatagggc ggcgcctaca atccatgcca acccgttcca
     3181 tgtgctcgcc gaggcggcat aaatcgccgt gacgatcagc ggtccaatga tcgaagttag
     3241 gctggtaaga gccgcgagcg atccttgaag ctgtccctga tggtcgtcat ctacctgcct
     3301 ggacagcatg gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa gaatcataat
     3361 ggggaaggcc atccagcctc gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc
     3421 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     3481 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     3541 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     3601 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     3661 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgac gctctccctt
     3721 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     3781 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
     3841 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
     3901 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
     3961 cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact
     4021 atagggagac cacaacggtt tccctctagt gccggctccg gagagctctt taattaagcg
     4081 gccgccctgc aggactcgag ttctagaaat aattttgttt aactttaaga aggagatata
     4141 catatgaaat cttctcacca tcaccatcac catggttctt ctatgaaaat cgaagaaggt
     4201 aaactggtaa tctggattaa cggcgataaa ggctataacg gtctcgctga agtcggtaag
     4261 aaattcgaga aagataccgg aattaaagtc accgttgagc atccggataa actggaagag
     4321 aaattcccac aggttgcggc aactggcgat ggccctgaca ttatcttctg ggcacacgac
     4381 cgctttggtg gctacgctca atctggcctg ttggctgaaa tcaccccgga caaagcgttc
     4441 caggacaagc tgtatccgtt tacctgggat gccgtacgtt acaacggcaa gctgattgct
     4501 tacccgatcg ctgttgaagc gttatcgctg atttataaca aagatctgct gccgaacccg
     4561 ccaaaaacct gggaagagat cccggcgctg gataaagaac tgaaagcgaa aggtaagagc
     4621 gcgctgatgt tcaacctgca agaaccgtac ttcacctggc cgctgattgc tgctgacggg
     4681 ggttatgcgt tcaagtatga aaacggcaag tacgacatta aagacgtggg cgtggataac
     4741 gctggcgcga aagcgggtct gaccttcctg gttgacctga ttaaaaacaa acacatgaat
     4801 gcagacaccg attactccat cgcagaagct gcctttaata aaggcgaaac agcgatgacc
     4861 atcaacggcc cgtgggcatg gtccaacatc gacaccagca aagtgaatta tggtgtaacg
     4921 gtactgccga ccttcaaggg tcaaccatcc aaaccgttcg ttggcgtgct gagcgcaggt
     4981 attaacgccg ccagtccgaa caaagagctg gcaaaagagt tcctcgaaaa ctatctgctg
     5041 actgatgaag gtctggaagc ggttaataaa gacaaaccgc tgggtgccgt agcgctgaag
     5101 tcttacgagg aagagttggc gaaagatcca cgtattgccg ccactatgga aaacgcccag
     5161 aaaggtgaaa tcatgccgaa catcccgcag atgtccgctt tctggtatgc cgtgcgtact
     5221 gcggtgatca acgccgccag cggtcgtcag actgtcgatg aagccctgaa agacgcgcag
     5281 actaattcga gctcgaacaa caacaacaat aacaataaca acaacctcgg gatcgaggaa
     5341 aacctgtact tccaatccaa tgctatgatt cttccgtctt tccccgactt aacaggattg
     5401 gttgtcaact tgaagttcac ggcccgcgca gagttcagtt taaaccacga aatggctgtg
     5461 gatgcattct tacgtcactc tttaaatttg ggcgagagct actcgcatca cctttcaatc
     5521 attacgccag aaaatggccg cttattttat cgcgaaggcg atacgtaccg tttcgtagtc
     5581 atcgcgatgg gaaaccaaca acaaaccaac tccatttggc acacacttat caatcacttg
     5641 cgcaaactgc ctgatagcgc tccaatcacg gataagcagg caccgctgcg taacaatatt
     5701 aagcttgaat ccttaaatga tctgtttgat gggattcctg tgtcgagtaa ggaatcactg
     5761 gacgcttata ctcttcaacg cgcgatggag caggggctgg catggcacaa agccgcgaac
     5821 cttactgaac agccactgga catccagtgg tattggcaat caaccgtccg tattcttcat
     5881 gccgatcaca aacagcacaa aggggaacaa cgctactgcc gtgatgcggt acaactgaca
     5941 cctttgcttc tgttaaagcg catctatgag acattgaaca atgtggctac ctatttcgga
     6001 cttaaaacta ataaaaatac aacagaaaac caccaagcct ggctgaagga acaggcccag
     6061 tacatcgaaa tccaacaccc ggacctttac tggattgata cgccctattt cggcaaggac
     6121 gctgaaaaaa acacactggg tggtatggcc ggaaatttca cgctgtcgtt aaaacccggt
     6181 atcgaacccg gacttttagc aatgctgatc cttacacaga tggtcggcgt gggccaacgt
     6241 cgcacttcgg gactgggtaa atactggttg aaacactcat taaaacacgc acacttaatt
     6301 ttaggtttaa agcctaatcg cgtgactcgt tcgcagacgc ttctggactg tattatccaa
     6361 ccccacatca ttagtcaagc aatcgcagag atcgaaaaga agacgaatat tgatacctta
     6421 aacgaacgca cgttaagcca ggttcagtca gcgattggtc aattgcgtaa gcatcagtac
     6481 caggccccaa agttacaggg ctttactatt ccaaaaaagg atggaactga acgccttttg
     6541 gcagtgagtc cgttgtatga ccgcatcttg cagaaagcgg cagccatcgt cttaacacct
     6601 gggctggacg ccatcatgtc gcaggcgagc tacggttatc gtaaggggct tagtcgccag
     6661 caggtccgtt atgagattca gaacgcatac cgtcagggtt atcactgggt gtatgaaagc
     6721 gatattgaag acttcttcga cgccgtctac cgtccccagc ttattaaccg tcttaaaagt
     6781 ttattgggaa acgatccact gtgggaacag atcgagtcat ggctggggca ggacattcat
     6841 attaaggaca ccatcattga gcgtacgcct aatttaggat tgccccaggg aagcccatta
     6901 agtccgttat tagctaactt tatcttggat gacttcgact cagatttaga aacgcatggg
     6961 ttcaagatca tccgttttgc tgacgatttc attattttat gtaagtcaca acatgaagcc
     7021 caacaggctg cgcacgcggt cgaacagtcg ttaaaagagg tcaagctgtc aatcaatgtt
     7081 gaaaagacgc acatcattca gctgaaccaa gggttccgct ttttaggtta cttgttccgc
     7141 gaggaccacg cgatcgagat cgcaggggaa aagtccgacg gccgcacaac ttttgcggcc
     7201 gagcagaccc caacgaatct gcctccgtgg ttagctaatc tgggtacaaa atcaccgcag
     7261 cctttggccg acgatgacct tccgaaaaag tcctacggtc agatcgaaac gcaaggaaca
     7321 cacttagtct tagcgggcga tgcccaaatt atcacgacag ataaccaaaa cttgatcgtc
     7381 aagaaagatg acaaaatcac tcataagatc tcgctggagc agttacacgc ggtaacgttg
     7441 atcggtttac acactatgac tttaccggct aaacaccgtc tgctggagca caagattcca
     7501 gtccacatcg cagaccgtac tgggcgttat ttgggtgcgg tgacaagttt ccaaccggct
     7561 caaaacaact acaaaaattg gttcattcaa ctgcagatgt gtgatcgcga acccttcgct
     7621 cacgctatcg cgcagcagat cgtaattagt cgtattcaca accaacgcca gacgctgtta
     7681 aaacgtaaag ctcatcgcaa gcagctgcaa cagacactta gcaatctgaa gaaattgcaa
     7741 tacaaggtca cggctgctac gaaacgctct agtctgaatg gtttagaagg cagtgctact
     7801 cgcgaatact ttcagcaatt taatctgttt cttcctgagt gggcccattt ttcgaagcgc
     7861 acacgtcgcc ctcctaaaga tcctttcaat gtgctgctga gcctggggta cacgattctg
     7921 tattctcaca cagacgccat cttacagtcg gctgggttta tcacatggaa aggcatctat
     7981 caccagcagt cggccgctca tgcagccttg gcctccgata ttatggaatc gtatcgccac
     8041 ttagtagaac gttatgccat ctacattatt aatcacggtc aaattaaaca agatgatttt
     8101 cgccaggaaa aagaccactt gggtcaagac acgatccgtc tgtccgctga ggcccgccgc
     8161 cgctatgttg gggggcttat taaccgtttc cagaagttct cgaaagataa gacccttcat
     8221 caacatctgt accagcaagc tcagcaatta aagaacgcga tgcacaatca acagtcatcc
     8281 cagtttcaag tatggaagga gttgaagtga attggaagtg gataacggat ccgcgatcgc
     8341 ggcgcgccac ctggtggccg gccggtacca cgcgtgcgcg ctgatccggc tgctaacaaa
     8401 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     8461 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggatat
     8521 ccacaggacg ggtgtggtcg ccatgatcgc gtagtcgata gtggctccaa gtagcgaagc
     8581 gagcaggact gggcggcggc caaagcggtc ggacagtgct ccgagaacgg gtgcgcatag
     8641 aaattgcatc aacgcatata gcgctagcag cacgccatag tgactggcga tgctgtcgga
     8701 atggacgata tcccgcaaga ggcccggcag taccggcata accaagccta tgcctacagc
     8761 atccagggtg acggtgccga ggatgacgat gagcgcattg ttagatttca tacacggtgc
     8821 ctgactgcgt tagcaattta actgtgataa actaccgcat taaagcttat cgatgataag
     8881 ctgtcaaaca tgagaa
//
