LOCUS       sequence-449593-        7206 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid codes for the Slug-KH nickase with a sg(AGC)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-sgAGC-SlugD10A-3XFLAG-SV40 NLS-ITR2
FEATURES             Location/Qualifiers
     source          1..7206
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1530..1733)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1559..1579)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1734..2037
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_RNA        complement(2133..2208)
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     promoter        complement(2236..2326)
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     complement(2296..2315)
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     repeat_region   2359..2499
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2359..2488
                     /label=AAV2 ITR
     primer_bind     complement(2536..2553)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2536..2552)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2545..2567)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2761..2780)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2880..2902
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2940..2958)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3026..3130
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3131..3199,3200..3991)
                     /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(3349..3368)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4162..4750
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4651..4670
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    5038..5059
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5074..5079,5080..5097,5098..5104)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5112..5128
                     /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     5117..5139
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5136..5152
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5136..5152
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5210..5350
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5221..5350
                     /label=AAV2 ITR
     polyA_signal    5367..5415
                     /note="synthetic polyadenylation signal"
     CDS             complement(5434..5454)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(5458..5529)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
ORIGIN
        1 ctcattgatg aacttctgct tgtccttgct attattttct ctggccagct cgatgatgat
       61 atcttcaggc acgccgtact tttctatgat cttgttaata aggttaatgg cctggccgaa
      121 ggtcctcttc accacagggc tcaggatgaa ctcgtcgatc atagccttcg ggatcttgtt
      181 ggcggctgtc aggttgatct tcttaggctt aatgttcagg tgggcgaaga tttccatctg
      241 ggcccggcta gaataccact gctcttccag gaccagccgg atgcatttca ggctgagccg
      301 gtgggtcccg gtgtagccag tcagctgggc gatgttttcc ttatcttcct cgttcagcag
      361 gatgtccagt tccgtcagct tagatttgat gctatccttg tcctggtaga tggtcaggat
      421 ctcagcgatc tggtccagca cgtcttcgtt ctccaggatg ctctggtcaa acagcacgct
      481 tttcaggtcg tggtagagct taaactcggt aaactggggc ttgccagact ttgtgattct
      541 gtagcccttg atatcttcag ggttgacatt gatctcattg gcgatctgct tcagtgtagg
      601 tttcttcttc tgcttgaaca cgttctcgat gatgtgatac ttctcatgat actcgagctt
      661 agacagtcca tctctttgga tgaccaggtt gttcaggtcg ttcagagcgt tgaacaggtc
      721 ggcactgtag gcgtatttca cgctggccag ctcatctggg aagtaggtgc agtggcccat
      781 cagtgtctcg taccaagcct tagggtcgcc ttcccagcca tatggactgc ccttgccggg
      841 gccctcgaag tattcccgtc tcatctcaac cagttcgatg tacttgttga tgaagttctc
      901 gtccagctgg tggaagttct tctgcacatt cagcagctgg atgatttcct tgatgatatc
      961 agcggtcttg aaccggtttt tctctccccg cacctggccc tcattcattc tttccagctg
     1021 gatctggcac acaaatttgt ctttcagcag cttgctgttc ttattcagct gttccttggt
     1081 gctgagctcg ttccccacgt cgtcgttgct gtcgatcacg tcgatcttgt ggatgcctct
     1141 tctcttggcg atatggagca gggcaatcac gagttcgtcc ttgctcaggg cttcagacag
     1201 gcccttcacg cggatggcgt aggggtttgt agactgagga atctgggact gatccagcag
     1261 attataatcc tcgagcagct tcttcacccg ttcaagtctg tgaatgcggc gtcgcttcag
     1321 tcttctgctt ccacgtttgg accttcttcc ctcgttgttt tccacgttgg cctcggggaa
     1381 cagtctaacg ccggcatcga tgatattctt tgtctcgtag tcgatcaggc cgtagcccac
     1441 gctggtgatt ccgatggcca gtcccagtat gaatttttgg ttcatggtgg cgaccggtag
     1501 cgctagcgga tctgacggtt cactaaacca gctctgctta tatagacctc ccaccgtaca
     1561 cgcctaccgc ccatttgcgt caatggggcg gagttgttac gacattttgg aaagtcccgt
     1621 tgattttggt gccaaaacaa actcccattg acgtcaatgg ggtggagact tggaaatccc
     1681 cgtgagtcaa accgctatcc acgcccattg atgtactgcc aaaaccgcat caccatggta
     1741 atagcgatga ctaatacgta gatgtactgc caagtaggaa agtcccataa ggtcatgtac
     1801 tgggcataat gccaggcggg ccatttaccg tcattgacgt caataggggg cgtacttggc
     1861 atatgataca cttgatgtac tgccaagtgg gcagtttacc gtaaatactc cacccattga
     1921 cgtcaatgga aagtccctat tggcgttact atgggaacat acgtcattat tgacgtcaat
     1981 gggcgggggt cgttgggcgg tcagccaggc gggccattta ccgtaagtta tgtaacgcgg
     2041 aactccatat atgggctatg aactaatgac cccgtaattg attactatta ataactacct
     2101 aggcgggccc cggtaccctc tagaaaaaaa aatctcgcca acaagttgac gagataaaca
     2161 cggcattttg ccttgtttta gtagattctg tttccagagt actaaaacgc tgctgctgct
     2221 gctgctcggg aaagagtggt ctcatacaga acttataaga ttcccaaatc caaagacatt
     2281 tcacgtttat ggtgatttcc cagaacacat agcgacatgc aaatatacgc ctagcctagc
     2341 ctacctaggg ggggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg
     2401 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg
     2461 cggcctcagt gagcgagcga gcgcgcagct gcctgcagga tctagatgca ttcgcgaggt
     2521 accgagctcg aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     2581 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     2641 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat
     2701 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag
     2761 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     2821 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     2881 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     2941 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     3001 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     3061 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     3121 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     3181 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     3241 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     3301 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     3361 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     3421 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     3481 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     3541 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     3601 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     3661 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     3721 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     3781 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     3841 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     3901 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     3961 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     4021 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     4081 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     4141 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     4201 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     4261 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     4321 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     4381 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4441 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4501 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4561 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4621 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4681 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4741 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4801 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     4861 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     4921 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     4981 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     5041 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     5101 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
     5161 cgccaagctt gcatgcaggc ctctgcagtc gacgggcccg ggatccgatc ctgcaggcag
     5221 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
     5281 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
     5341 aggggttcct gcggccggaa ttcttacaca caaaaaacca acacacagat ctaatgaaaa
     5401 taaagatctt ttattgaatt cttagctggc ctccaccttc ctcttcttct tggggggctt
     5461 atcgtcgtca tctttgtaat ccttatcatc atcgtcctta tagtccttgt cgtcgtcgtc
     5521 cttgtagtca tttcctcgct tgaacagcag ctgaggtttt gtgtactggg tgttggtgaa
     5581 cacgttgccc agcacgtcgg ttgtcagttt ttcgatgcta ttcacctttt tgccgatggt
     5641 ctttttgatg tgaggttcgc ccttgatatt gttcagctcg cagtattctt tgtatctgat
     5701 gtcaggcaga tccagttcga tcatgtttct ggtgtcgcta ttcacgccga tgatcttgta
     5761 gatctcgccg tccagcttga tcagatcatt cttgtagaag ctagcgataa acttggcgtt
     5821 tttgtcgatg gccttgccca gtttgagctt gtcgtacttc tgttcgggta tgtagtagta
     5881 attgtctttc ttcagcacgt ccaggtagct gatggtgatg aacttatatc ccttgtcggt
     5941 caggtagacg tcgaatctat aaggcttgat gctcagcttc accaacttct tggtgctgct
     6001 cttgaactgg tgggtcacat ctaggtggct gcccagcttg tttccaatgt acttcaggct
     6061 tttcacgata gggccgttgt tttttttgct gtatttggtc aggtactcgc ctgtttcctc
     6121 gtggtacttg gccagaggat tcttctcatt agcgtactgc ttcatgatca cttccaactt
     6181 ctcgaaggtc cgcggatcgt gttggtacat gaggaacttc tcgggtgact tgtcaaactg
     6241 cttcttcagg gtggtattat ctttggcata gatatctttg atggtctgca cgatgtaggt
     6301 ggagttgtcc ttcttccggg tgctgtacag tgtatcgttg atcagctttc tgttaggctt
     6361 cttgtccact ctgtggctgt acttgaagtt tctgaagtcc ttgatgtcct ggacctgctt
     6421 agggatgatg aacatttcgc tgtagttatc ctcgctatcc acctggatat ccagctgctt
     6481 gctctcgatc tcaggttttt ccagcacaga gttcacggct ttcagcttct tgttctcttt
     6541 gaacaggaaa tcggcattag cgataatcag ggcgtcctcg gcgtggtgct tgtagccgtg
     6601 gttccgttcc ttcttaaact tccacacttt ccgcaggtaa tcggtgaagc tgccgttgat
     6661 cgttttcacc ttcacgttca tattgttggc gctgaagtag gccttcaggt agtttgtcag
     6721 ctcggctgtg gcgtaccggg tatccaccag gtttctattg atgaactctt tttgcacctc
     6781 gaacttgttg atgtcccgtt cttccagcag atactctttc ttttttttgc tgattctatc
     6841 ctgggacttg ctcaggttca ggatgtgttg tttaaactgg ttgtaagaga gcttggactt
     6901 gccgctgtta aagtactggt aaggggtcag gttggatttt ttggagttct cgctctgctt
     6961 cacgagcact ttgttgtggt aagagttgtc gaaggacaca gatcttggaa tgatgtggtc
     7021 cacctcgtag tggttggggt tgttcagcag gtcctccaga gggatgcttt ccagtgagta
     7081 caggcacttg ccctcctgct cgtcgtgcag tctaatcttc tcgaccagtc tcttggcgtt
     7141 ctggttgcca tacttgccga tgatttcgtt aattctcttt ctggtgttct cgttcttttt
     7201 ctgcat
//
