LOCUS       sequence-491099-        7537 bp    DNA     circular SYN 30-OCT-2025
DEFINITION  Bacterial expression of ECOR31 2TMbeta residues 72-200 with 
            N-terminal His-SUMO tag and co-expression of ECOR31 mCpol.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7537)
  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 7537)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 30, 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_126
FEATURES             Location/Qualifiers
     source          1..7537
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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             42..64
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        826..844
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    845..869
                     /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)."
     CDS             930..2030
                     /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     2004..2027
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2085..2105
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2486..2530
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2568..2586)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2582..2629
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2666..3121
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2753..2772)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2963..2984
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3236..4027,4028..4096))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3859..3878
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4097..4189)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4270..4858
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4759..4778
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5012..5029
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5044..5183
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5109..5128)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5269..5291
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5285..5476)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6048..7130)
                     /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     7092..7111
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7131..7208)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7414..7433
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(7521..7537,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgggagtcaa
      121 gactgagaac aacgatcata ttaatttgaa ggtggcgggg caggatggtt ctgtggtgca
      181 gtttaagatt aagaggcata caccacttag taaactaatg aaagcctatt gtgaacgaca
      241 gggattgtca atgaggcaga tcagattccg atttgacggg caaccaatca atgaaacaga
      301 cacacctgca cagttggaaa tggaggatga agatacaatt gatgtgtttc aacagcagac
      361 gggtggatca ttcaagaaag acatatatcc tgacttaaat ggaatttggg ttggacatat
      421 aatcacagaa acaaatgatg aatttgaagt ccgcgcaaaa ataaagcaag cactccttgt
      481 aacaaagata gaaatgcacg gccccacagt taaatctgtt acgctagaag caacgccaac
      541 taaggaactg gataacaaca aattgtatta tgtatataaa tcaaccccta aaaaccctag
      601 ttggagcgag tatattggct cgactatatt cgacgtaatt gaaagtaaca atgcgctcca
      661 actttccggt cgatattata cagacagaaa atccgtgggg agaatatcaa taaaaagaat
      721 atcacttaac actgactccg atatatcttt ttattaactt gcggccgcat aatgcttaag
      781 tcgaacagaa agtaatcgta ttgtacacgg ccgcataatc gaaattaata cgactcacta
      841 taggggaatt gtgagcggat aacaattccc catcttagta tattagttaa gtataagaag
      901 gagatataca tatggcagat ctcaattgta tgaaaatcga agaaggtaaa ctggtaatct
      961 ggattaacgg cgataaaggc tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag
     1021 ataccggaat taaagtcacc gttgagcatc cggataaact ggaagagaaa ttcccacagg
     1081 ttgcggcaac tggcgatggc cctgacatta tcttctgggc acacgaccgc tttggtggct
     1141 acgctcaatc tggcctgttg gctgaaatca ccccggacaa agcgttccag gacaagctgt
     1201 atccgtttac ctgggatgcc gtacgttaca acggcaagct gattgcttac ccgatcgctg
     1261 ttgaagcgtt atcgctgatt tataacaaag atctgctgcc gaacccgcca aaaacctggg
     1321 aagagatccc ggcgctggat aaagaactga aagcgaaagg taagagcgcg ctgatgttca
     1381 acctgcaaga accgtacttc acctggccgc tgattgctgc tgacgggggt tatgcgttca
     1441 agtatgaaaa cggcaagtac gacattaaag acgtgggcgt ggataacgct ggcgcgaaag
     1501 cgggtctgac cttcctggtt gacctgatta aaaacaaaca catgaatgca gacaccgatt
     1561 actccatcgc agaagctgcc tttaataaag gcgaaacagc gatgaccatc aacggcccgt
     1621 gggcatggtc caacatcgac accagcaaag tgaattatgg tgtaacggta ctgccgacct
     1681 tcaagggtca accatccaaa ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca
     1741 gtccgaacaa agagctggca aaagagttcc tcgaaaacta tctgctgact gatgaaggtc
     1801 tggaagcggt taataaagac aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag
     1861 agttggcgaa agatccacgt attgccgcca ctatggaaaa cgcccagaaa ggtgaaatca
     1921 tgccgaacat cccgcagatg tccgctttct ggtatgccgt gcgtactgcg gtgatcaacg
     1981 ccgccagcgg tcgtcagact gtcgatgaag ccctgaaaga cgcgcagact aattcgagct
     2041 cgaacaacaa caacaataac aataacaaca acctcgggat cgaggaaaac ctgtacttcc
     2101 aatccaatgc aatgtacata gcgatagatg gcgatgatgt ggggagaaaa ataacctcat
     2161 catacttatc caactcagaa gagaggttga catacataag caacaaatta aatgatacca
     2221 caaaaaaaat atctaaaatg cttttatcca atggatttga gataattttc caagccgcag
     2281 atggtgttac agcgaaaaca gacaacgagg tcaatcttaa ctttgttttt gataagataa
     2341 aatcctattc ctttgacgaa attacatttt cagctggtgt gggagcgaat ctaagagaag
     2401 cctatgttgc actgttaaat tctaaaagca acggtaagaa tatgatttct atttacaaag
     2461 acatattata accctcgagt ctggtaaaga aaccgctgct gcgaaatttg aacgccagca
     2521 catggactcg tctactagcg cagcttaatt aacctaggct gctgccaccg ctgagcaata
     2581 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
     2641 aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2701 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     2761 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     2821 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     2881 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     2941 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     3001 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     3061 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     3121 tctggcggca cgatggcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     3181 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     3241 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     3301 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     3361 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     3421 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     3481 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     3541 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     3601 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     3661 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     3721 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     3781 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     3841 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     3901 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     3961 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     4021 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     4081 aatgttgaat actcatactc ttcctttttc aatcatgatt gaagcattta tcagggttat
     4141 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggtcatgac
     4201 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4261 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     4321 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     4381 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     4441 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     4501 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     4561 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     4621 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     4681 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     4741 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     4801 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     4861 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     4921 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     4981 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     5041 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
     5101 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
     5161 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
     5221 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     5281 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     5341 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     5401 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
     5461 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
     5521 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
     5581 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
     5641 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
     5701 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
     5761 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
     5821 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
     5881 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgctagtca
     5941 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     6001 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     6061 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     6121 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
     6181 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
     6241 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
     6301 tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta
     6361 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
     6421 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
     6481 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
     6541 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
     6601 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
     6661 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
     6721 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
     6781 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
     6841 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
     6901 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
     6961 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
     7021 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
     7081 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc
     7141 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg
     7201 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag
     7261 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc
     7321 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat
     7381 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc
     7441 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat
     7501 cgatctcgat cccgcgaaat taatacgact cactata
//
