LOCUS       sequence-488899-        6266 bp    DNA     circular SYN 09-OCT-2025
DEFINITION  ECOR31 mCpol bacterial expression under T7 promoter, C-term 
            TEV-MBP-HIS10 .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6266)
  AUTHORS   Doherty EE, Adler BA, Yoon PH, Hsieh K, Loi K, Armbuster EG, Lahiri 
            A, Bolling CS, Wilcox XE, Akkati A, Iavarone AT, Pogliano J, Doudna 
            JA
  TITLE     A miniature CRISPR-Cas10 enzyme confers immunity by an inverse 
            signaling pathway.
  JOURNAL   bioRxiv [Preprint]. 2025 Apr 1:2025.03.28.646030. doi: 
            10.1101/2025.03.28.646030.
  PUBMED    40196485
REFERENCE   2  (bases 1 to 6266)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 9, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2025 Apr 1:2025.03.28.646030. doi: 
            10.1101/2025.03.28.646030."
COMMENT     Sequence Label: ED_p_060
FEATURES             Location/Qualifiers
     source          1..6266
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2..21
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2..20
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             109..131
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             496..516
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             532..1632
                     /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     1606..1629
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1642..1671
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(1797..1815)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1811..1858
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2224..2229,2230..2246,2247..2252))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2279..2297)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2365..2469
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2470..2538,2539..3330)
                     /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(2688..2707)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3501..4089
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3990..4009
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4243..4260
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4275..4414
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4340..4359)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4500..4522
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4516..4707)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     6165..6184
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ttaatacgac tcactatagg gagaccacaa cggtttccct ctagtgccgg ctccggagag
       61 ctctttaatt aagcggccgc cctgcaggac tcgagttcta gaaataattt tgtttaactt
      121 taagaaggag atatacatat gtacatagcg atagatggcg atgatgtggg gagaaaaata
      181 acctcatcat acttatccaa ctcagaagag aggttgacat acataagcaa caaattaaat
      241 gataccacaa aaaaaatatc taaaatgctt ttatccaatg gatttgagat aattttccaa
      301 gccgcagatg gtgttacagc gaaaacagac aacgaggtca atcttaactt tgtttttgat
      361 aagataaaat cctattcctt tgacgaaatt acattttcag ctggtgtggg agcgaatcta
      421 agagaagcct atgttgcact gttaaattct aaaagcaacg gtaagaatat gatttctatt
      481 tacaaagaca tattagaaaa cctgtacttc caatccggct ctagcggctc tatgaaaatc
      541 gaagaaggta aactggtaat ctggattaac ggcgataaag gctataacgg tctcgctgaa
      601 gtcggtaaga aattcgagaa agataccgga attaaagtca ccgttgagca tccggataaa
      661 ctggaagaga aattcccaca ggttgcggca actggcgatg gccctgacat tatcttctgg
      721 gcacacgacc gctttggtgg ctacgctcaa tctggcctgt tggctgaaat caccccggac
      781 aaagcgttcc aggacaagct gtatccgttt acctgggatg ccgtacgtta caacggcaag
      841 ctgattgctt acccgatcgc tgttgaagcg ttatcgctga tttataacaa agatctgctg
      901 ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa
      961 ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact tcacctggcc gctgattgct
     1021 gctgacgggg gttatgcgtt caagtatgaa aacggcaagt acgacattaa agacgtgggc
     1081 gtggataacg ctggcgcgaa agcgggtctg accttcctgg ttgacctgat taaaaacaaa
     1141 cacatgaatg cagacaccga ttactccatc gcagaagctg cctttaataa aggcgaaaca
     1201 gcgatgacca tcaacggccc gtgggcatgg tccaacatcg acaccagcaa agtgaattat
     1261 ggtgtaacgg tactgccgac cttcaagggt caaccatcca aaccgttcgt tggcgtgctg
     1321 agcgcaggta ttaacgccgc cagtccgaac aaagagctgg caaaagagtt cctcgaaaac
     1381 tatctgctga ctgatgaagg tctggaagcg gttaataaag acaaaccgct gggtgccgta
     1441 gcgctgaagt cttacgagga agagttggcg aaagatccac gtattgccgc cactatggaa
     1501 aacgcccaga aaggtgaaat catgccgaac atcccgcaga tgtccgcttt ctggtatgcc
     1561 gtgcgtactg cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa
     1621 gacgcgcaga ctggttcttc tcaccatcac catcaccatc accatcacca ttaacattgg
     1681 aagtggataa cggatccgcg atcgcggcgc gccacctggt ggccggccgg taccacgcgt
     1741 gcgcgctgat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     1801 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     1861 gaaaggagga actatatccg gatatccaca ggacgggtgt ggtcgccatg atcgcgtagt
     1921 cgatagtggc tccaagtagc gaagcgagca ggactgggcg gcggccaaag cggtcggaca
     1981 gtgctccgag aacgggtgcg catagaaatt gcatcaacgc atatagcgct agcagcacgc
     2041 catagtgact ggcgatgctg tcggaatgga cgatatcccg caagaggccc ggcagtaccg
     2101 gcataaccaa gcctatgcct acagcatcca gggtgacggt gccgaggatg acgatgagcg
     2161 cattgttaga tttcatacac ggtgcctgac tgcgttagca atttaactgt gataaactac
     2221 cgcattaaag cttatcgatg ataagctgtc aaacatgaga attcttgaag acgaaagggc
     2281 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     2341 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     2401 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata acattgaaaa
     2461 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     2521 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     2581 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     2641 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     2701 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     2761 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     2821 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     2881 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     2941 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     3001 accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     3061 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     3121 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     3181 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     3241 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     3301 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     3361 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     3421 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     3481 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     3541 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     3601 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     3661 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     3721 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     3781 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     3841 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     3901 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     3961 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     4021 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     4081 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     4141 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     4201 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     4261 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     4321 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     4381 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     4441 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     4501 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     4561 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     4621 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     4681 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     4741 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     4801 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     4861 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     4921 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     4981 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     5041 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     5101 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     5161 catgcgcacc cgtggccagg acccaacgct gcccgagatg cgccgcgtgc ggctgctgga
     5221 gatggcggac gcgatggata tgttctgcca agggttggtt tgcgcattca cagttctccg
     5281 caagaattga ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt gccgccggct
     5341 tccattcagg tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg gaggcagaca
     5401 aggtataggg cggcgcctac aatccatgcc aacccgttcc atgtgctcgc cgaggcggca
     5461 taaatcgccg tgacgatcag cggtccaatg atcgaagtta ggctggtaag agccgcgagc
     5521 gatccttgaa gctgtccctg atggtcgtca tctacctgcc tggacagcat ggcctgcaac
     5581 gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa tggggaaggc catccagcct
     5641 cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg
     5701 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     5761 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     5821 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     5881 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     5941 gggttgaagg ctctcaaggg catcggtcga cgctctccct tatgcgactc ctgcattagg
     6001 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     6061 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     6121 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     6181 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     6241 aggatcgaga tctcgatccc gcgaaa
//
