LOCUS       sequence-449590-        7206 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid codes for the Slug-KH nickase with a sg(GCA)6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7206)
  AUTHORS   Mangin A, Dion V, Menzies G
  TITLE     Developing small Cas9 hybrids using molecular modeling
  JOURNAL   bioRxiv 2023.10.24.563270
REFERENCE   2  (bases 1 to 7206)
  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: "bioRxiv 
            2023.10.24.563270"
COMMENT     Sequence Label: pUC57-ITR2-H1-sgGCA-SlugD10A-3XFLAG-SV40 NLS-ITR2
FEATURES             Location/Qualifiers
     source          1..7206
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1601..1804)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1630..1650)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1805..2108
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_RNA        complement(2204..2279)
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     promoter        complement(2307..2397)
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     complement(2367..2386)
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     repeat_region   2430..2570
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2430..2559
                     /label=AAV2 ITR
     primer_bind     complement(2607..2624)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2607..2623)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2616..2638)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2832..2851)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2951..2973
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3011..3029)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3097..3201
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3202..3270,3271..4062)
                     /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     complement(3420..3439)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4233..4821
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4722..4741
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    5109..5130
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5145..5150,5151..5168,5169..5175)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5183..5199
                     /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     5188..5210
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5207..5223
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5207..5223
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5281..5421
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5292..5421
                     /label=AAV2 ITR
     polyA_signal    5438..5486
                     /note="synthetic polyadenylation signal"
     CDS             complement(5505..5525)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(5529..5600)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
ORIGIN
        1 gcgttctggt tgccatactt gccgatgatt tcgttaattc tctttctggt gttctcgttc
       61 tttttctgca tctcattgat gaacttctgc ttgtccttgc tattattttc tctggccagc
      121 tcgatgatga tatcttcagg cacgccgtac ttttctatga tcttgttaat aaggttaatg
      181 gcctggccga aggtcctctt caccacaggg ctcaggatga actcgtcgat catagccttc
      241 gggatcttgt tggcggctgt caggttgatc ttcttaggct taatgttcag gtgggcgaag
      301 atttccatct gggcccggct agaataccac tgctcttcca ggaccagccg gatgcatttc
      361 aggctgagcc ggtgggtccc ggtgtagcca gtcagctggg cgatgttttc cttatcttcc
      421 tcgttcagca ggatgtccag ttccgtcagc ttagatttga tgctatcctt gtcctggtag
      481 atggtcagga tctcagcgat ctggtccagc acgtcttcgt tctccaggat gctctggtca
      541 aacagcacgc ttttcaggtc gtggtagagc ttaaactcgg taaactgggg cttgccagac
      601 tttgtgattc tgtagccctt gatatcttca gggttgacat tgatctcatt ggcgatctgc
      661 ttcagtgtag gtttcttctt ctgcttgaac acgttctcga tgatgtgata cttctcatga
      721 tactcgagct tagacagtcc atctctttgg atgaccaggt tgttcaggtc gttcagagcg
      781 ttgaacaggt cggcactgta ggcgtatttc acgctggcca gctcatctgg gaagtaggtg
      841 cagtggccca tcagtgtctc gtaccaagcc ttagggtcgc cttcccagcc atatggactg
      901 cccttgccgg ggccctcgaa gtattcccgt ctcatctcaa ccagttcgat gtacttgttg
      961 atgaagttct cgtccagctg gtggaagttc ttctgcacat tcagcagctg gatgatttcc
     1021 ttgatgatat cagcggtctt gaaccggttt ttctctcccc gcacctggcc ctcattcatt
     1081 ctttccagct ggatctggca cacaaatttg tctttcagca gcttgctgtt cttattcagc
     1141 tgttccttgg tgctgagctc gttccccacg tcgtcgttgc tgtcgatcac gtcgatcttg
     1201 tggatgcctc ttctcttggc gatatggagc agggcaatca cgagttcgtc cttgctcagg
     1261 gcttcagaca ggcccttcac gcggatggcg taggggtttg tagactgagg aatctgggac
     1321 tgatccagca gattataatc ctcgagcagc ttcttcaccc gttcaagtct gtgaatgcgg
     1381 cgtcgcttca gtcttctgct tccacgtttg gaccttcttc cctcgttgtt ttccacgttg
     1441 gcctcgggga acagtctaac gccggcatcg atgatattct ttgtctcgta gtcgatcagg
     1501 ccgtagccca cgctggtgat tccgatggcc agtcccagta tgaatttttg gttcatggtg
     1561 gcgaccggta gcgctagcgg atctgacggt tcactaaacc agctctgctt atatagacct
     1621 cccaccgtac acgcctaccg cccatttgcg tcaatggggc ggagttgtta cgacattttg
     1681 gaaagtcccg ttgattttgg tgccaaaaca aactcccatt gacgtcaatg gggtggagac
     1741 ttggaaatcc ccgtgagtca aaccgctatc cacgcccatt gatgtactgc caaaaccgca
     1801 tcaccatggt aatagcgatg actaatacgt agatgtactg ccaagtagga aagtcccata
     1861 aggtcatgta ctgggcataa tgccaggcgg gccatttacc gtcattgacg tcaatagggg
     1921 gcgtacttgg catatgatac acttgatgta ctgccaagtg ggcagtttac cgtaaatact
     1981 ccacccattg acgtcaatgg aaagtcccta ttggcgttac tatgggaaca tacgtcatta
     2041 ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg cgggccattt accgtaagtt
     2101 atgtaacgcg gaactccata tatgggctat gaactaatga ccccgtaatt gattactatt
     2161 aataactacc taggcgggcc ccggtaccct ctagaaaaaa aaatctcgcc aacaagttga
     2221 cgagataaac acggcatttt gccttgtttt agtagattct gtttccagag tactaaaact
     2281 gctgctgctg ctgctgccgg gaaagagtgg tctcatacag aacttataag attcccaaat
     2341 ccaaagacat ttcacgttta tggtgatttc ccagaacaca tagcgacatg caaatatacg
     2401 cctagcctag cctacctagg gggggccgca ggaaccccta gtgatggagt tggccactcc
     2461 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
     2521 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg atctagatgc
     2581 attcgcgagg taccgagctc gaattcactg gccgtcgttt tacaacgtcg tgactgggaa
     2641 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     2701 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     2761 tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg
     2821 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     2881 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     2941 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     3001 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     3061 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     3121 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     3181 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     3241 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     3301 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     3361 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     3421 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     3481 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     3541 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     3601 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     3661 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     3721 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     3781 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     3841 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     3901 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     3961 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     4021 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     4081 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     4141 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     4201 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     4261 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     4321 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     4381 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     4441 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     4501 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     4561 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     4621 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     4681 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     4741 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     4801 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     4861 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     4921 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     4981 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg
     5041 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     5101 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
     5161 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
     5221 gaccatgatt acgccaagct tgcatgcagg cctctgcagt cgacgggccc gggatccgat
     5281 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
     5341 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
     5401 actccatcac taggggttcc tgcggccgga attcttacac acaaaaaacc aacacacaga
     5461 tctaatgaaa ataaagatct tttattgaat tcttagctgg cctccacctt cctcttcttc
     5521 ttggggggct tatcgtcgtc atctttgtaa tccttatcat catcgtcctt atagtccttg
     5581 tcgtcgtcgt ccttgtagtc atttcctcgc ttgaacagca gctgaggttt tgtgtactgg
     5641 gtgttggtga acacgttgcc cagcacgtcg gttgtcagtt tttcgatgct attcaccttt
     5701 ttgccgatgg tctttttgat gtgaggttcg cccttgatat tgttcagctc gcagtattct
     5761 ttgtatctga tgtcaggcag atccagttcg atcatgtttc tggtgtcgct attcacgccg
     5821 atgatcttgt agatctcgcc gtccagcttg atcagatcat tcttgtagaa gctagcgata
     5881 aacttggcgt ttttgtcgat ggccttgccc agtttgagct tgtcgtactt ctgttcgggt
     5941 atgtagtagt aattgtcttt cttcagcacg tccaggtagc tgatggtgat gaacttatat
     6001 cccttgtcgg tcaggtagac gtcgaatcta taaggcttga tgctcagctt caccaacttc
     6061 ttggtgctgc tcttgaactg gtgggtcaca tctaggtggc tgcccagctt gtttccaatg
     6121 tacttcaggc ttttcacgat agggccgttg ttttttttgc tgtatttggt caggtactcg
     6181 cctgtttcct cgtggtactt ggccagagga ttcttctcat tagcgtactg cttcatgatc
     6241 acttccaact tctcgaaggt ccgcggatcg tgttggtaca tgaggaactt ctcgggtgac
     6301 ttgtcaaact gcttcttcag ggtggtatta tctttggcat agatatcttt gatggtctgc
     6361 acgatgtagg tggagttgtc cttcttccgg gtgctgtaca gtgtatcgtt gatcagcttt
     6421 ctgttaggct tcttgtccac tctgtggctg tacttgaagt ttctgaagtc cttgatgtcc
     6481 tggacctgct tagggatgat gaacatttcg ctgtagttat cctcgctatc cacctggata
     6541 tccagctgct tgctctcgat ctcaggtttt tccagcacag agttcacggc tttcagcttc
     6601 ttgttctctt tgaacaggaa atcggcatta gcgataatca gggcgtcctc ggcgtggtgc
     6661 ttgtagccgt ggttccgttc cttcttaaac ttccacactt tccgcaggta atcggtgaag
     6721 ctgccgttga tcgttttcac cttcacgttc atattgttgg cgctgaagta ggccttcagg
     6781 tagtttgtca gctcggctgt ggcgtaccgg gtatccacca ggtttctatt gatgaactct
     6841 ttttgcacct cgaacttgtt gatgtcccgt tcttccagca gatactcttt cttttttttg
     6901 ctgattctat cctgggactt gctcaggttc aggatgtgtt gtttaaactg gttgtaagag
     6961 agcttggact tgccgctgtt aaagtactgg taaggggtca ggttggattt tttggagttc
     7021 tcgctctgct tcacgagcac tttgttgtgg taagagttgt cgaaggacac agatcttgga
     7081 atgatgtggt ccacctcgta gtggttgggg ttgttcagca ggtcctccag agggatgctt
     7141 tccagtgagt acaggcactt gccctcctgc tcgtcgtgca gtctaatctt ctcgaccagt
     7201 ctcttg
//
