LOCUS       sequence-235845-        8630 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses E. coli codon optimized Cas12i1 effector in the pET28a 
            backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8630)
  AUTHORS   Yan WX, Hunnewell P, Alfonse LE, Carte JM, Keston-Smith E, 
            Sothiselvam S, Garrity AJ, Chong S, Makarova KS, Koonin EV, Cheng 
            DR, Scott DA
  TITLE     Functionally diverse type V CRISPR-Cas systems.
  JOURNAL   Science. 2019 Jan 4;363(6422):88-91. doi: 10.1126/science.aav7271. 
            Epub 2018 Dec 6.
  PUBMED    30523077
REFERENCE   2  (bases 1 to 8630)
  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"; doi: 
            "10.1126/science.aav7271"; journalName: "Science"; date: "2019-01-4-
            4"; volume: "363"; issue: "6422"; pages: "88-91"
COMMENT     Sequence Label: pET28a-mH6-Cas12i1
FEATURES             Location/Qualifiers
     source          1..8630
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2..24
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(18..209)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1018..2100)
                     /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     2062..2081
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2101..2178)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2384..2403
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2487..2506
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2487..2505
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2506..2530
                     /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)."
     promoter        join(2539..2544,2545..2562,2563..2568)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     RBS             2588..2610
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2642..2659
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6051..6069)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6065..6112
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      6149..6604
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6236..6255)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6446..6467
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6696..7511)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(6696..7511)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     7419..7438
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7503..7522
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      7633..8221
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8122..8141
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8375..8392
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    8407..8546
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(8472..8491)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
       61 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
      121 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
      181 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
      241 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
      301 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
      361 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
      421 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
      481 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
      541 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
      601 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
      661 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
      721 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
      781 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
      841 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
      901 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
      961 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     1021 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     1081 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     1141 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     1201 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     1261 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     1321 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     1381 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     1441 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     1501 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     1561 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     1621 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     1681 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     1741 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     1801 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     1861 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     1921 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     1981 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     2041 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     2101 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     2161 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     2221 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     2281 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     2341 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     2401 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     2461 ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
     2521 taacaattcc cctctagatt tacactttat gcttccggct cgtatgtttc tagaaagctt
     2581 aaataatttt gtttaacttt aagaaggaga tttaaatatg aaaatcgaag aaggtaaagg
     2641 tcaccatcac catcaccaca tgagcaacaa agaaaagaac gcgagcgaaa cccgtaaggc
     2701 gtacaccacc aaaatgatcc cgcgtagcca cgaccgtatg aagctgctgg gcaactttat
     2761 ggactatctg atggatggca ccccgatttt ctttgaactg tggaaccagt tcggtggcgg
     2821 tatcgaccgt gatatcatta gcggcaccgc gaacaaagat aagattagcg acgatctgct
     2881 gctggcggtt aactggttta aagtgatgcc gatcaacagc aaaccgcaag gcgtgagccc
     2941 gagcaacctg gcgaacctgt tccagcaata cagcggtagc gaaccggaca tccaggcgca
     3001 agagtatttc gcgagcaact ttgacaccga aaagcaccag tggaaagata tgcgtgttga
     3061 gtacgaacgt ctgctggcgg agctgcaact gagccgtagc gacatgcacc atgatctgaa
     3121 gctgatgtac aaagaaaagt gcattggcct gagcctgagc accgcgcact atatcaccag
     3181 cgtgatgttt ggcaccggtg cgaagaacaa ccgtcagacc aaacatcaat tctatagcaa
     3241 agtgattcag ctgctggagg aaagcaccca gatcaacagc gttgagcaac tggcgagcat
     3301 cattctgaaa gcgggtgact gcgatagcta ccgtaaactg cgtatccgtt gcagccgtaa
     3361 gggtgcgacc ccgagcattc tgaaaatcgt gcaagactat gaactgggca ccaaccacga
     3421 cgatgaggtg aacgttccga gcctgattgc gaacctgaaa gaaaagctgg gccgttttga
     3481 gtacgaatgc gagtggaagt gcatggagaa aatcaaggcg ttcctggcga gcaaagttgg
     3541 cccgtactat ctgggtagct atagcgcgat gctggagaac gcgctgagcc cgattaaggg
     3601 tatgaccacc aagaactgca aattcgtgct gaaacagatt gacgcgaaga acgatatcaa
     3661 atacgaaaac gagccgtttg gcaagatcgt tgaaggtttc tttgacagcc cgtatttcga
     3721 gagcgatacc aacgtgaaat gggttctgca cccgcaccat attggcgaaa gcaacatcaa
     3781 gaccctgtgg gaggacctga acgcgattca tagcaaatac gaggaagaca tcgcgagcct
     3841 gagcgaagat aagaaagaga agcgtatcaa ggtttaccag ggcgatgtgt gccaaaccat
     3901 caacacctat tgcgaggaag tgggcaagga agcgaaaacc ccgctggttc agctgctgcg
     3961 ttacctgtat agccgtaaag acgatattgc ggtggacaag atcattgatg gtatcacctt
     4021 cctgagcaag aaacacaagg tggagaaaca gaagattaac ccggttatcc aaaaataccc
     4081 gagcttcaac tttggcaaca acagcaagct gctgggtaaa atcattagcc cgaaagacaa
     4141 gctgaaacat aacctgaagt gcaaccgtaa ccaagtggat aactacatct ggattgaaat
     4201 caaggttctg aacaccaaga ccatgcgttg ggagaaacac cattatgcgc tgagcagcac
     4261 ccgttttctg gaggaagttt actatccggc gaccagcgaa aacccgccgg atgcgctggc
     4321 ggcgcgtttc cgtaccaaaa ccaacggtta tgagggcaag ccggcgctga gcgcggaaca
     4381 gattgagcaa atccgtagcg cgccggttgg cctgcgtaaa gtgaagaaac gtcagatgcg
     4441 tctggaagcg gcgcgtcagc aaaacctgct gccgcgttat acctggggca aggattttaa
     4501 cattaacatc tgcaaacgtg gtaacaactt cgaagttacc ctggcgacca aggtgaagaa
     4561 aaagaaagag aagaactaca aagtggttct gggttatgac gcgaacattg tgcgtaaaaa
     4621 cacctatgcg gcgattgagg cgcatgcgaa cggtgatggt gtgattgact ataacgatct
     4681 gccggttaag ccgatcgaaa gcggctttgt gaccgttgag agccaggttc gtgacaaaag
     4741 ctacgatcaa ctgagctata acggtgtgaa gctgctgtac tgcaaaccgc atgttgaaag
     4801 ccgtcgtagc ttcctggaga aatatcgtaa cggcaccatg aaggacaacc gtggtaacaa
     4861 cattcagatc gacttcatga aggattttga agcgatcgcg gacgatgaaa ccagcctgta
     4921 ctattttaac atgaagtact gcaaactgct gcagagcagc attcgtaacc acagcagcca
     4981 agcgaaagaa tatcgtgagg aaatcttcga gctgctgcgt gatggcaagc tgagcgtgct
     5041 gaaactgagc agcctgagca acctgagctt cgtgatgttt aaggttgcga aaagcctgat
     5101 tggcacctac tttggtcatc tgctgaagaa accgaagaac agcaaaagcg acgttaaggc
     5161 tccgccgatc accgacgaag ataagcagaa agcggacccg gagatgttcg cgctgcgtct
     5221 ggcgctggag gaaaaacgtc tgaacaaggt taaaagcaag aaagaagtga ttgcgaacaa
     5281 gatcgttgcg aaagcgctgg agctgcgtga caaatacggc ccggtgctga ttaagggcga
     5341 gaacatcagc gataccacca agaaaggtaa gaaaagcagc accaacagct tcctgatgga
     5401 ctggctggcg cgtggcgtgg cgaacaaggt taaagaaatg gtgatgatgc accagggtct
     5461 ggagttcgtt gaggtgaacc cgaactttac cagccaccaa gatccgttcg ttcataagaa
     5521 cccggagaac acctttcgtg cgcgttacag ccgttgcacc ccgagcgaac tgaccgagaa
     5581 gaaccgtaaa gaaatcctga gcttcctgag cgacaagccg agcaaacgtc cgaccaacgc
     5641 gtactataac gagggtgcga tggcgtttct ggcgacctat ggcctgaaga aaaacgacgt
     5701 gctgggtgtt agcctggaaa agttcaaaca gattatggcg aacatcctgc atcagcgtag
     5761 cgaggatcaa ctgctgtttc cgagccgtgg cggtatgttc tacctggcga cctataagct
     5821 ggacgcggat gcgaccagcg ttaactggaa cggcaaacag ttttgggtgt gcaacgcgga
     5881 cctggttgcg gcgtacaacg tgggtctggt tgacatccaa aaagatttca agaaaaagta
     5941 ataaaagctt ctcgagagag accatggcca ccggtctcta tcgaagatat gaggagctca
     6001 aactcgagct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     6061 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     6121 aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     6181 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     6241 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     6301 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     6361 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     6421 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     6481 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     6541 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     6601 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     6661 tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa
     6721 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     6781 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     6841 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     6901 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt
     6961 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
     7021 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga
     7081 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     7141 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     7201 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
     7261 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     7321 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     7381 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     7441 tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga
     7501 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
     7561 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     7621 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     7681 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     7741 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     7801 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     7861 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     7921 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     7981 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     8041 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     8101 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     8161 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     8221 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     8281 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     8341 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     8401 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     8461 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     8521 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     8581 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
//
