LOCUS       sequence-235829-        7798 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses E. coli codon optimized Cas12g1 effector in the pET28a 
            backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7798)
  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 7798)
  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-Cas12g1
FEATURES             Location/Qualifiers
     source          1..7798
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    775..914
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(840..859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1000..1022
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1016..1207)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2016..3098)
                     /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     3060..3079
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3099..3176)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3382..3401
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3485..3504
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3485..3503
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3504..3528
                     /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(3537..3542,3543..3560,3561..3566)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     RBS             3586..3608
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3640..3657
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6217..6235)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6231..6278
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      6315..6770
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6402..6421)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6612..6633
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6862..7677)
                     /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(6862..7677)
                     /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     7585..7604
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7669..7688
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
      841 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
      901 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
      961 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     1021 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     1081 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     1141 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     1201 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     1261 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     1321 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     1381 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     1441 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     1501 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     1561 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     1621 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     1681 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     1741 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     1801 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     1861 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     1921 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     1981 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     2041 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2101 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     2161 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     2221 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     2281 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     2341 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     2401 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     2461 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     2521 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     2581 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     2641 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     2701 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     2761 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     2821 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     2881 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     2941 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     3001 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     3061 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     3121 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     3181 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     3241 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     3301 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     3361 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     3421 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     3481 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagattta
     3541 cactttatgc ttccggctcg tatgtttcta gaaagcttaa ataattttgt ttaactttaa
     3601 gaaggagatt taaatatgaa aatcgaagaa ggtaaaggtc accatcacca tcaccacatg
     3661 gcgcaggcga gcagcacccc ggcggtgagc ccgcgtccgc gtccgcgtta ccgtgaggaa
     3721 cgtaccctgg ttcgtaaact gctgccgcgt ccgggtcaaa gcaagcagga gttccgtgaa
     3781 aacgtgaaga aactgcgtaa ggcgttcctg cagtttaacg cggatgtgag cggtgtttgc
     3841 caatgggcga tccagttccg tccgcgttat ggcaaaccgg cggagccgac cgaaaccttt
     3901 tggaagttct ttctggagcc ggaaaccagc ctgccgccga acgacagccg tagcccggag
     3961 ttccgtcgtc tgcaggcgtt tgaagcggcg gcgggtatta acggtgcggc ggcgctggac
     4021 gatccggcgt tcaccaacga gctgcgtgat agcattctgg cggtggcgag ccgtccgaag
     4081 accaaagaag cgcaacgtct gtttagccgt ctgaaagact accagccggc gcaccgtatg
     4141 atcctggcga aggttgcggc ggagtggatt gaaagccgtt accgtcgtgc gcatcagaac
     4201 tgggagcgta actatgagga atggaagaaa gaaaagcaag agtgggaaca gaaccacccg
     4261 gagctgaccc cggagatccg tgaagcgttc aaccaaattt ttcagcaact ggaagtgaag
     4321 gaaaaacgtg ttcgtatctg cccggcggcg cgtctgctgc aaaacaaaga caactgccag
     4381 tatgcgggca agaacaaaca cagcgtgctg tgcaaccaat tcaacgagtt taagaaaaac
     4441 catctgcagg gcaaggcgat caagttcttt tacaaggatg cggaaaagta tctgcgttgc
     4501 ggtctgcaaa gcctgaaacc gaacgttcag ggcccgttcc gtgaggactg gaacaagtac
     4561 ctgcgttata tgaacctgaa agaggaaacc ctgcgtggca agaacggtgg ccgtctgccg
     4621 cactgcaaaa acctgggcca agagtgcgaa tttaacccgc ataccgcgct gtgcaagcag
     4681 taccagcaac agctgagcag ccgtccggac ctggtgcaac acgatgaact gtaccgtaaa
     4741 tggcgtcgtg agtattggcg tgaaccgcgt aagccggtgt tccgttaccc gagcgttaaa
     4801 cgtcatagca tcgcgaagat ttttggcgag aactatttcc aggcggactt taagaacagc
     4861 gtggttggtc tgcgtctgga tagcatgccg gcgggccagt acctggagtt cgcgtttgcg
     4921 ccgtggccgc gtaactatcg tccgcaaccg ggcgaaaccg aaatcagcag cgtgcacctg
     4981 catttcgttg gcacccgtcc gcgtattggc ttccgttttc gtgttccgca caaacgtagc
     5041 cgttttgact gcacccagga agagctggat gagctgcgta gccgtacctt cccgcgtaaa
     5101 gcgcaagatc agaagtttct ggaggcggcg cgtaagcgtc tgctggaaac cttcccgggt
     5161 aacgcggagc aggaactgcg tctgctggcg gtggacctgg gcaccgatag cgcgcgtgcg
     5221 gcgttcttta tcggcaaaac cttccaacag gcgtttccgc tgaagatcgt taaaattgag
     5281 aagctgtacg aacagtggcc gaaccaaaaa caagcgggcg accgtcgtga tgcgagcagc
     5341 aagcaaccgc gtccgggtct gagccgtgac cacgtgggcc gtcatctgca aaaaatgcgt
     5401 gcgcaggcga gcgagattgc gcaaaagcgt caagaactga ccggcacccc ggcgccggaa
     5461 accaccaccg accaagcggc gaagaaagcg accctgcagc cgttcgatct gcgtggcctg
     5521 accgttcaca ccgcgcgtat gatccgtgac tgggcgcgtc tgaacgcgcg tcaaatcatt
     5581 cagctggcgg aggaaaacca ggtggatctg attgttctgg aaagcctgcg tggtttccgt
     5641 ccgccgggtt acgagaacct ggaccaagaa aagaaacgtc gtgtggcgtt ctttgcgcat
     5701 ggtcgtatcc gtcgtaaagt gaccgagaag gcggttgaac gtggcatgcg tgtggttacc
     5761 gtgccgtatc tggcgagcag caaagtttgc gcggagtgcc gtaagaaaca aaaagataac
     5821 aagcagtggg agaagaacaa gaaacgtggt ctgttcaagt gcgagggttg cggcagccaa
     5881 gcgcaggttg acgaaaacgc ggcgcgtgtg ctgggtcgtg tgttctgggg tgagatcgaa
     5941 ctgccgaccg cgattccgta ataaaagctt cgacccttga cggaactggc cgatttgcta
     6001 aaatattttt gatgcttact tagtcatctg gttggcaaac ctccgcggac cttcgggacc
     6061 aatggagagg aacccagccg agaagcatcg agccggtaaa tgccggaaat ttttgactcg
     6121 agagagacca tggccaccgg tctctatcga agatatgagg agctcaaact cgagctgcta
     6181 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     6241 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     6301 gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     6361 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     6421 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     6481 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     6541 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     6601 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     6661 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     6721 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     6781 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     6841 tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt
     6901 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
     6961 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac
     7021 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     7081 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt
     7141 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     7201 accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg
     7261 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     7321 attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc
     7381 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     7441 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     7501 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat
     7561 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     7621 catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac
     7681 accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt
     7741 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttc
//
