LOCUS       sequence-375575-        9198 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PET28-FnCas12a-EP16 can efficiently recognize a broad range of PAM 
            sequences including YN (Y = C or T), TAC and CAA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9198)
  AUTHORS   Wang L, Wang H, Liu H, Zhao Q, Liu B, Wang L, Zhang J, Zhu J, Bao R,
            Luo Y
  TITLE     Improved CRISPR-Cas12a-assisted one-pot DNA editing method enables 
            seamless DNA editing.
  JOURNAL   Biotechnol Bioeng. 2019 Jun;116(6):1463-1474. doi: 
            10.1002/bit.26938. Epub 2019 Feb 11.
  PUBMED    30730047
REFERENCE   2  (bases 1 to 9198)
  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: "10.1002/bit.26938";
            journalName: "Biotechnol Bioeng"; date: "2019-06"; volume: "116"; 
            issue: "6"; pages: "1463-1474"
COMMENT     Sequence Label: PET28-FnCas12a-EP16
FEATURES             Location/Qualifiers
     source          1..9198
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4058..4078)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(4100..4117)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             4139..4161
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4176..4200
                     /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)."
     primer_bind     complement(4200..4219)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4201..4219)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4303..4322)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4528..4605
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4606..5688
                     /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     complement(4625..4644)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             6497..6688
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(6682..6704)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6790..6929
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6845..6864
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6944..6961)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7115..7703)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7195..7214)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(7814..7833)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             7825..8640
                     /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
                     TAFQVLEEYPDSGENIVDALVVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(7898..7917)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(8732..9187)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8869..8890)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     9081..9100
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcac tcgagtcaat tgttgcggtt ctgcacgaac tcaaagtact
      181 cctcgttctt gatgaccagg ttcagcttct tgccctcctg attgttcttg atcctgccca
      241 gcagcatcag gcccttcagg ccgatgtgat aggcgccgtt ggcgtcggca tcctgaggca
      301 tattcttggg ggcctgtctg ctgtcgaaga agttgccgtt cacatcggcc acgggggaga
      361 tcaggtagtc cagctctgtg ccggtcttgc tgttccgcat ctgcaggatt gtattcagca
      421 cggaggtcag cttggcgaaa aacttcttat cgctctcgcc acagatggcg gccttgatgc
      481 actcgccgtg gccatactcg atgctgtagt ccttcagcag cttctccagc tcctttgttg
      541 ggtacacctc ccgggtgtcc caattgtggt tcttatcgga atttctaaag ttgatcagcc
      601 ggctgccgaa agaggcgatg gtccacttgc ccttggcggc cttgtcgcca aagttcttat
      661 aatcgaagga aaactcgaag tagcccttgt ccagattata acagatctta tcgaacttgc
      721 tgaaaaactc ctggctctta gacacggact catacttagg gtacagctgg ttcacaaagc
      781 ctgtcacggg gcagatcttg gaggtgaagc cggctggcac atagtagatg atgcctgtct
      841 gcttgcccat cttcttgaat gtctcaaagg gggcggtcag ctggtatgcc ctcagcacgc
      901 ctccggtctt gtcgaactcg ttatccttaa acaccaggta attcagcttc tcgatcagca
      961 tcttctccag cttctgatac acctgcttct ccaccttaaa gcggcccctc ttaaagccga
     1021 agttcagatc ctcgaacacc acgatggcat tgtactcgat gaccagcttg gcgatctcgt
     1081 gcaccacctg agacagatag ccctccttca tctccttgat attgttgatc ttcttccagt
     1141 ccttgcgggc ggagtcccta tccttctcga tggcggccag cttatcgtgg tagtttgtct
     1201 tcatcctgtc attgccgatg atgttgaagg tatcctgctt gatgatattg cccttgccat
     1261 ccaccagggt atagtaggcc aggtgtctct cgccccggtc gatgctcagg atgtgcacat
     1321 cgttggcctt ctccttcagc agcagattga tctcgtcatt aaacttgttg gcgccagagg
     1381 acttgaagtt gattgtgatt ggacagtgga aaaagaactt atcctcggtg aaccgcttgt
     1441 ccttgatcag atcgtactcg aacacgctct ccttcttagg attgtcctta ttcttgttgg
     1501 cgatggcctc cttggctggg tgtgtgatct tcttagggat ggactgcttc ctgtaaaaca
     1561 gctcggcctc gccgttcagc ttatacacca cgtcctgcag atttctctca tcgaacaggg
     1621 ccttccagta cagggtgtgc aggtttggcc ggcccttgga ataggcgctg aagtccttgt
     1681 tatagatctg gaacaggtac agcttgccct gattcaccac gctgtcgatg tagctctcag
     1741 agatgttctc aaatgtcagc ttatagccct gattctccac ctctctgtag aactcgtcga
     1801 tggagttgta ccgctgggtg tcgctaaacc tgaagccaaa atccttccac tcagggtgct
     1861 tggagatgct ctgcttgtag aagtcgataa acttacggca atcctcgata ttgaactcaa
     1921 acttctcata gcccttctgg ggagagccgt tctttgtgtg ggtggagtga tttctgatcc
     1981 gcaggatgtc ctcggatggg ttgtagaact tgatagactt ggcggaaaag aacaccttag
     2041 gcagcatctt attggcgccg ggcagcagcc tatacacgat cttcttgtag ccctcgccct
     2101 tgttctcctt gatggccttg tcatcgaaga tcttattgtt cttcttattc atcacgccca
     2161 gatagtactt gtcatccttg atgaacagga tggctgttct gttaggctcc acattcttat
     2221 cccagcctct ggccagggtg ctgttctcga agttcagctt aaacttctcg tcggagtaag
     2281 gcttctgtgt gatataattt ctgatcttgt tgtacagggg cacgatgttg gccagctcga
     2341 agtaacactc ctcgaacacc agataaaagt gctcgtcctt atccaggata ttggccttat
     2401 cctcagactg ggagatgtgg aagatcttca gcttgtgcag cagattgttg gtctggtcca
     2461 gcagatcctt gatggccttc acgtcatcct cggcagaggc ctgcagcagg tccttcttgc
     2521 cctggttctg gtacttgatg gagatctggg ccagattgtc cttgttctgg gcgatctcat
     2581 caaagatcat ggggatggcg gcgaagttgg ccaggatctc ctcaaatctg cactgcttgt
     2641 cgatatcccg gtgcttattg aactcctcca gggccagctt gattgtctcc aggctcagat
     2701 acttggcctt ctcggtcttc ttggcgatca gctcctgctc cttcttagag ggattatcca
     2761 ggttctttgg ggcgatctgc tgtgtgatgt actccagcac ggcggtgccg atcacgctat
     2821 agtcatcaaa cacctgctga gacaggtcgg tcagggactt atcgttcttg aagtagatct
     2881 tagacaggtc cagcttctgg gccttcaggt catcgaacag caggctcagt gtctccttga
     2941 tagacttctc ctccacggtc ttgaaggcgg cgatctgctc ataaaagctc tgcattgtgg
     3001 tcaccacgtc agagtcatcc tccagcttat cgatcacaaa gctcttagac tctgtatcgg
     3061 acaggatctg cttgaacagc acgctcatct tgtacttctt cagggtcttg tcgttgatct
     3121 gctggctata cagattgatg tactcgttga tgccctttct cttggtattc tcgccgttca
     3181 caaacttgcc gccgatgatt gtattgaact tggtgatgcc ggactggttc aggtaattgt
     3241 tgaaattggc gatctcaaac acctcgtcca gagaaaacac ccgctggttc acctcggatg
     3301 tcttatagtc gatatcgaag gtcagctcct cggccagatc cttcttgatc tgctcgtaat
     3361 tgatggcctc gggggccttg tccttcaggg actcatactt ggccttgttc tccaggaact
     3421 ttggcagatt gtcatccacg atgcggtaga tgatgcttgt agggatgtcg ttgctgctgt
     3481 acacgttctt cctgttctcg tggaagccgc taaagtatgt ggtccagccc ttaaagctct
     3541 tgatgatctc cagggcctcg tcgatatcgg tgatatcaga gttggccttg aacagctcga
     3601 tgccattgtc cttagactgc ttcagccaca ggatcaggtc ggactcctgg cccttcttgg
     3661 catcgatcag attctggttg aacagattct taaacttctc ggagtccttg atatactcgc
     3721 tgatctgctt cttgatggtg tccttggcgc tcttgaagtc cttctgtagg ttgtcatcgt
     3781 cagacttctt cagcttgaaa tacacgtcgc tgtaattctg cagcagatcc tcagagatgc
     3841 acacgctgct caggatctcc tcgataaaga actggtggta cttatcgatg atctgcttgg
     3901 ccttcttata gtccttggcg cgcttctcat cgtccaggat caggcccctg gccttgatgt
     3961 tctccagtgt cttgccctgg gggatcagct caaaccgcag ggtcttggac aggctgtact
     4021 tattcacgaa ctcctgatag atgctcatat ccatggcgcc ctgaaaatac aggttttcgg
     4081 tcgttgggat atcgtaatcg tgatggtgat ggtgatggta gtacgaggac atggtatatc
     4141 tccttcttaa agttaaacaa aattatttct agaggggaat tgttatccgc tcacaattcc
     4201 cctatagtga gtcgtattaa tttcgcggga tcgagatctc gatcctctac gccggacgca
     4261 tcgtggccgg catcaccggc gccacaggtg cggttgctgg cgcctatatc gccgacatca
     4321 ccgatgggga agatcgggct cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta
     4381 tggtggcagg ccccgtggcc gggggactgt tgggcgccat ctccttgcat gcaccattcc
     4441 ttgcggcggc ggtgctcaac ggcctcaacc tactactggg ctgcttccta atgcaggagt
     4501 cgcataaggg agagcgtcga gatcccggac accatcgaat ggcgcaaaac ctttcgcggt
     4561 atggcatgat agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg
     4621 ttatacgatg tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac
     4681 caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg
     4741 aattacattc ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc
     4801 gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct
     4861 cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa
     4921 gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac
     4981 tatccgctgg atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg
     5041 ttatttcttg atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac
     5101 ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta
     5161 gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc
     5221 actcgcaatc aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt
     5281 tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc
     5341 aacgatcaga tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt
     5401 gcggatatct cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg
     5461 ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg
     5521 caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa
     5581 agaaaaacca ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     5641 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     5701 taatgtaagt tagctcactc attaggcacc gggatctcga ccgatgccct tgagagcctt
     5761 caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac
     5821 tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgctctggg tcattttcgg
     5881 cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat
     5941 cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa
     6001 gcaggccatt atcgccggca tggcggcccc acgggtgcgc atgatcgtgc tcctgtcgtt
     6061 gaggacccgg ctaggctggc ggggttgcct tactggttag cagaatgaat caccgatacg
     6121 cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa caacatgaat
     6181 ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat
     6241 tatgttccgg atctgcatcg caggatgctg ctggctaccc tgtggaacac ctacatctgt
     6301 attaacgaag cgctggcatt gaccctgagt gatttttctc tggtcccgcc gcatccatac
     6361 cgccagttgt ttaccctcac aacgttccag taaccgggca tgttcatcat cagtaacccg
     6421 tatcgtgagc atcctctctc gtttcatcgg tatcattacc cccatgaaca gaaatccccc
     6481 ttacacggag gcatcagtga ccaaacagga aaaaaccgcc cttaacatgg cccgctttat
     6541 cagaagccag acattaacgc ttctggagaa actcaacgag ctggacgcgg atgaacaggc
     6601 agacatctgt gaatcgcttc acgaccacgc tgatgagctt taccgcagct gcctcgcgcg
     6661 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     6721 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     6781 gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac
     6841 tatgcggcat cagagcagat tgtactgaga gtgcaccatt gcggtgtgaa ataccgcaca
     6901 gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc
     6961 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     7021 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     7081 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     7141 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     7201 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     7261 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     7321 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     7381 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     7441 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     7501 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     7561 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     7621 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     7681 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     7741 agtggaacga aaactcacgt taagggattt tggtcatgaa caataaaact gtctgcttac
     7801 ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc ttgctctagg
     7861 ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat
     7921 gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc gccagagttg
     7981 tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta
     8041 aactggctga cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat
     8101 gatgcatggt tactcaccac tgcgatcccc gggaaaacag cattccaggt attagaagaa
     8161 tatcctgatt caggtgaaaa tattgttgat gcgctggtag tgttcctgcg ccggttgcat
     8221 tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg
     8281 caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc
     8341 tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca
     8401 gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata
     8461 ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta
     8521 tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt
     8581 attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa
     8641 gaattaattc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     8701 tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa
     8761 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     8821 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     8881 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     8941 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     9001 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     9061 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     9121 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     9181 ggcgcgtccc attcgcca
//
