LOCUS       sequence-418048-        8082 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express ApmFnuc protein with sumotag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8082)
  AUTHORS   Jiang K, Lim J, Sgrizzi S, Trinh M, Kayabolen A, Yutin N, Bao W, 
            Kato K, Koonin EV, Gootenberg JS, Abudayyeh OO
  TITLE     Programmable RNA-guided DNA endonucleases are widespread in 
            eukaryotes and their viruses.
  JOURNAL   Sci Adv. 2023 Sep 29;9(39):eadk0171. doi: 10.1126/sciadv.adk0171. 
            Epub 2023 Sep 27.
  PUBMED    37756409
REFERENCE   2  (bases 1 to 8082)
  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"; journalName: "Sci Adv."; 
            date: "2023-09-29"; volume: "9(39):eadk0171. doi"; pages: " 
            10.1126/sciadv.adk0171. Epub 2023 Sep 27"
COMMENT     Sequence Label: pDY1935
FEATURES             Location/Qualifiers
     source          1..8082
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     556..575
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(595..672)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     878..897
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        981..999
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1000..1024
                     /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             1039..1061
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1080..1097
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1107..1124
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(1137..1160,1161..1196,1197..1220)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             1221..1514
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(3127..3145)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3141..3188
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        3373..3391
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3392..3416
                     /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)."
     ncRNA           3718..3785
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     terminator      3786..3833
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(3854..3872)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3940..4044
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4045..4113,4114..4905)
                     /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(4263..4282)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5076..5664
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5565..5584
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5818..5835
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5850..5989
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5915..5934)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6075..6097
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6091..6282)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7594..8082,1..594))
                     /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"
ORIGIN
        1 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
       61 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
      121 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
      181 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
      241 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
      301 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
      361 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
      421 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
      481 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
      541 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
      601 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
      661 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
      721 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
      781 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
      841 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
      901 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
      961 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
     1021 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg ggcagcagcc
     1081 atcatcatca tcatcacagc agcggcctgg tgccgcgcgg cagccatatg gctagctgga
     1141 gccatccgca gtttgaaaaa ggtggtggta gcggtggtgg ttcaggtggt agtgcatggt
     1201 cacaccctca gtttgagaaa atgtcggact cagaagtcaa tcaagaagct aagccagagg
     1261 tcaagccaga agtcaagcct gagactcaca tcaatttaaa ggtgtccgat ggatcttcag
     1321 agatcttctt caagatcaaa aagaccactc ctttaagaag gctgatggaa gcgttcgcta
     1381 aaagacaggg taaggaaatg gactccttaa gattcttgta cgacggtatt agaatccaag
     1441 ctgatcagac ccctgaagat ttggacatgg aggataacga tattattgag gctcacagag
     1501 aacagattgg tggatccatg aaagaggcag ttaaaaatgt caaaccaaaa gttcccgcta
     1561 aaaagaggat tattactgga agtaaaacta agaaaaaggt ttttgtcaag aagaaaccac
     1621 ctgataagaa acctcttaag aaacccgtca aaaaaactgt taaaacatat aaactaaaat
     1681 ctatttatgt ttctaataaa gatcttaaaa tgtccaaatg gattcccact cctaaaaaag
     1741 aatttactga aattgaaact aacagttggt atgaacacag gaaatttgag aatcccaatg
     1801 gatctcctat acaatcctat aacaagattg tgcctgtagt tcctccggaa tctattaaac
     1861 aacagaattt agcaaacaaa cgtaagaaaa caaatagacc tattgttttt atttcttctg
     1921 agaaaatcag aatttatccc accaaagagc aacaaaaaat tttacaaact tggtttcgtt
     1981 tatttgcatg tatgtataat agttctattg attacattaa cagtaagaaa gttgttcttg
     2041 aatctggtag aattaatgtc gctgcaacca ggaaagtttg taataaaatt agcgttagaa
     2101 aagctctaaa aactattaga gacaatttaa tcaaatccac taatccatca ataatgaccc
     2161 atataatgga cgaagcgatt ggtttagctt gttcaaatta taagacatgt ttaaccaatt
     2221 atattgaagg tcaaattaaa aaatttgata ttaaaccttg gtctatctct aaaagacgaa
     2281 agattattgt cattgaacct ggatacttta aaggaaattc tttttgtccc acagtttttc
     2341 ccaaaatgaa atcttttaaa ccacttatta tgattgataa aactgtgact ttgcaatacg
     2401 attctgatac tagaaaatat atcttatttg tacctagagt cactcctaag tattctgtca
     2461 ataaggaaaa aaatagttgt ggtattgatc ctggtcttag agatttcctt acggtttatt
     2521 ctgaaaatga aactcaatca atttgtccta ttgaaatcgt agttaacacc actaaaaatg
     2581 aatacaagaa aattgataag atcaacgaaa tcatcaaaac taaaccaaat cttaatagta
     2641 aacgtaaaaa gaaacttaat agaggcttac gtaaatatca tagaagagtt actaataaga
     2701 tgaaagacat gcactataaa gtttctcatg aattagtaaa tacttttgat aaaatttgta
     2761 taggtaaact aaatgtaaag tctatcttat cgaaagccaa tactgttcta aaaagtgcct
     2821 tgaaaagaaa acttgcaact ctgagttttt ataggtttac tcagagatta actcatatgg
     2881 gttataagta tggcactgaa gtggttaatg tcaatgaata tttgaccaca aaaacctgct
     2941 caaactgcgg caagattaaa gatcttggag ctagcaagat ttacgaatgt gagtcttgtg
     3001 gaatgtacgc agatcgtgat gagaatgctg ccaagaatat cctaaaagta ggattaaagc
     3061 cttggtacaa acaaaaatag gcggccgcac tcgaggcccg aaaggaagct gagttggctg
     3121 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     3181 gttttttgct gaaaggagga actatatccg gatatcccgc aagaggcccg gcagtaccgg
     3241 cataaccaag cctatgccta cagcatccag ggtgacggtg ccgaggatga cgatgagcgc
     3301 attgttagat ttcatacacg gtgcctgact gcgttagcaa tttaactgtg ataaactacc
     3361 gcattaaagc tttaatacga ctcactatag gggaattgtg agcggataac aattccatga
     3421 gaatgctgcc aagaatatcc taaaagtagg attaaagcct tggtacaaac aaaaatagtc
     3481 taataaaatc aggggtacat tccgctagta ctccacccta cgggttaagc aaatgagaat
     3541 atcgaaacgg tatgcacagg attcttcgag tgataatctt aggatgactc actaaggaga
     3601 tgactaaagt gtatcattca atattgtatt gaacggtatt cttccataga gagttgattt
     3661 ttggagtatc cagaaatatc aactttttat gagcgggcag ccacctcctt gttattgggc
     3721 cggcatggtc ccagcctcct cgctggcgcc ggctgggcaa catgcttcgg catggcgaat
     3781 gggacctagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttggaattct
     3841 tgaagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     3901 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     3961 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     4021 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     4081 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     4141 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     4201 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     4261 ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact cggtcgccgc
     4321 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     4381 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     4441 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     4501 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     4561 aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta
     4621 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     4681 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     4741 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     4801 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     4861 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     4921 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     4981 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     5041 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     5101 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     5161 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     5221 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     5281 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     5341 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     5401 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     5461 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     5521 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     5581 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     5641 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     5701 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     5761 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     5821 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     5881 tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     5941 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     6001 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     6061 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     6121 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     6181 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     6241 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     6301 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     6361 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     6421 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     6481 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     6541 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     6601 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     6661 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     6721 cgacaggagc acgatcatgc gcacccgtgg ccaggaccca acgctgcccg agatgcgccg
     6781 cgtgcggctg ctggagatgg cggacgcgat ggatatgttc tgccaagggt tggtttgcgc
     6841 attcacagtt ctccgcaaga attgattggc tccaattctt ggagtggtga atccgttagc
     6901 gaggtgccgc cggcttccat tcaggtcgag gtggcccggc tccatgcacc gcgacgcaac
     6961 gcggggaggc agacaaggta tagggcggcg cctacaatcc atgccaaccc gttccatgtg
     7021 ctcgccgagg cggcataaat cgccgtgacg atcagcggtc caatgatcga agttaggctg
     7081 gtaagagccg cgagcgatcc ttgaagctgt ccctgatggt cgtcatctac ctgcctggac
     7141 agcatggcct gcaacgcggg catcccgatg ccgccggaag cgagaagaat cataatgggg
     7201 aaggccatcc agcctcgcgt cgcgaacgcc agcaagacgt agcccagcgc gtcggccgcc
     7261 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
     7321 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
     7381 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
     7441 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
     7501 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     7561 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     7621 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     7681 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     7741 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     7801 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     7861 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     7921 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     7981 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     8041 cggctgaatt tgattgcgag tgagatattt atgccagcca gc
//
