LOCUS       sequence-449592-        7205 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid codes for the Slug-KH nickase with a sg(CAG)6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7205)
  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 7205)
  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-sgCAG-SlugD10A-3XFLAG-SV40 NLS-ITR2
FEATURES             Location/Qualifiers
     source          1..7205
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   432..572
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   443..572
                     /label=AAV2 ITR
     promoter        605..695
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     616..635
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     misc_RNA        722..797
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     enhancer        893..1196
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1197..1400
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1351..1371
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             4607..4678
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     CDS             4682..4702
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4721..4769
                     /note="synthetic polyadenylation signal"
     repeat_region   4786..4926
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4786..4915
                     /label=AAV2 ITR
     primer_bind     complement(4984..5000)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4984..5000)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4997..5019)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5008..5024
                     /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        complement(join(5032..5038,5039..5056,5057..5062))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5077..5098
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5386..5974)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5466..5485)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6145..6936,6937..7005))
                     /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     6768..6787
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7006..7110)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7178..7196
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa
      421 tgcatctaga tcctgcaggc agctgcgcgc tcgctcgctc actgaggccg cccgggcaaa
      481 gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga
      541 gggagtggcc aactccatca ctaggggttc ctgcggcccc ccctaggtag gctaggctag
      601 gcgtatattt gcatgtcgct atgtgttctg ggaaatcacc ataaacgtga aatgtctttg
      661 gatttgggaa tcttataagt tctgtatgag accactcttt cccgcagcag cagcagcagc
      721 agttttagta ctctggaaac agaatctact aaaacaaggc aaaatgccgt gtttatctcg
      781 tcaacttgtt ggcgagattt ttttttctag agggtaccgg ggcccgccta ggtagttatt
      841 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
      901 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
      961 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     1021 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
     1081 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
     1141 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
     1201 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
     1261 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
     1321 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
     1381 aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagcgc taccggtcgc
     1441 caccatgaac caaaaattca tactgggact ggccatcgga atcaccagcg tgggctacgg
     1501 cctgatcgac tacgagacaa agaatatcat cgatgccggc gttagactgt tccccgaggc
     1561 caacgtggaa aacaacgagg gaagaaggtc caaacgtgga agcagaagac tgaagcgacg
     1621 ccgcattcac agacttgaac gggtgaagaa gctgctcgag gattataatc tgctggatca
     1681 gtcccagatt cctcagtcta caaaccccta cgccatccgc gtgaagggcc tgtctgaagc
     1741 cctgagcaag gacgaactcg tgattgccct gctccatatc gccaagagaa gaggcatcca
     1801 caagatcgac gtgatcgaca gcaacgacga cgtggggaac gagctcagca ccaaggaaca
     1861 gctgaataag aacagcaagc tgctgaaaga caaatttgtg tgccagatcc agctggaaag
     1921 aatgaatgag ggccaggtgc ggggagagaa aaaccggttc aagaccgctg atatcatcaa
     1981 ggaaatcatc cagctgctga atgtgcagaa gaacttccac cagctggacg agaacttcat
     2041 caacaagtac atcgaactgg ttgagatgag acgggaatac ttcgagggcc ccggcaaggg
     2101 cagtccatat ggctgggaag gcgaccctaa ggcttggtac gagacactga tgggccactg
     2161 cacctacttc ccagatgagc tggccagcgt gaaatacgcc tacagtgccg acctgttcaa
     2221 cgctctgaac gacctgaaca acctggtcat ccaaagagat ggactgtcta agctcgagta
     2281 tcatgagaag tatcacatca tcgagaacgt gttcaagcag aagaagaaac ctacactgaa
     2341 gcagatcgcc aatgagatca atgtcaaccc tgaagatatc aagggctaca gaatcacaaa
     2401 gtctggcaag ccccagttta ccgagtttaa gctctaccac gacctgaaaa gcgtgctgtt
     2461 tgaccagagc atcctggaga acgaagacgt gctggaccag atcgctgaga tcctgaccat
     2521 ctaccaggac aaggatagca tcaaatctaa gctgacggaa ctggacatcc tgctgaacga
     2581 ggaagataag gaaaacatcg cccagctgac tggctacacc gggacccacc ggctcagcct
     2641 gaaatgcatc cggctggtcc tggaagagca gtggtattct agccgggccc agatggaaat
     2701 cttcgcccac ctgaacatta agcctaagaa gatcaacctg acagccgcca acaagatccc
     2761 gaaggctatg atcgacgagt tcatcctgag ccctgtggtg aagaggacct tcggccaggc
     2821 cattaacctt attaacaaga tcatagaaaa gtacggcgtg cctgaagata tcatcatcga
     2881 gctggccaga gaaaataata gcaaggacaa gcagaagttc atcaatgaga tgcagaaaaa
     2941 gaacgagaac accagaaaga gaattaacga aatcatcggc aagtatggca accagaacgc
     3001 caagagactg gtcgagaaga ttagactgca cgacgagcag gagggcaagt gcctgtactc
     3061 actggaaagc atccctctgg aggacctgct gaacaacccc aaccactacg aggtggacca
     3121 catcattcca agatctgtgt ccttcgacaa ctcttaccac aacaaagtgc tcgtgaagca
     3181 gagcgagaac tccaaaaaat ccaacctgac cccttaccag tactttaaca gcggcaagtc
     3241 caagctctct tacaaccagt ttaaacaaca catcctgaac ctgagcaagt cccaggatag
     3301 aatcagcaaa aaaaagaaag agtatctgct ggaagaacgg gacatcaaca agttcgaggt
     3361 gcaaaaagag ttcatcaata gaaacctggt ggatacccgg tacgccacag ccgagctgac
     3421 aaactacctg aaggcctact tcagcgccaa caatatgaac gtgaaggtga aaacgatcaa
     3481 cggcagcttc accgattacc tgcggaaagt gtggaagttt aagaaggaac ggaaccacgg
     3541 ctacaagcac cacgccgagg acgccctgat tatcgctaat gccgatttcc tgttcaaaga
     3601 gaacaagaag ctgaaagccg tgaactctgt gctggaaaaa cctgagatcg agagcaagca
     3661 gctggatatc caggtggata gcgaggataa ctacagcgaa atgttcatca tccctaagca
     3721 ggtccaggac atcaaggact tcagaaactt caagtacagc cacagagtgg acaagaagcc
     3781 taacagaaag ctgatcaacg atacactgta cagcacccgg aagaaggaca actccaccta
     3841 catcgtgcag accatcaaag atatctatgc caaagataat accaccctga agaagcagtt
     3901 tgacaagtca cccgagaagt tcctcatgta ccaacacgat ccgcggacct tcgagaagtt
     3961 ggaagtgatc atgaagcagt acgctaatga gaagaatcct ctggccaagt accacgagga
     4021 aacaggcgag tacctgacca aatacagcaa aaaaaacaac ggccctatcg tgaaaagcct
     4081 gaagtacatt ggaaacaagc tgggcagcca cctagatgtg acccaccagt tcaagagcag
     4141 caccaagaag ttggtgaagc tgagcatcaa gccttataga ttcgacgtct acctgaccga
     4201 caagggatat aagttcatca ccatcagcta cctggacgtg ctgaagaaag acaattacta
     4261 ctacataccc gaacagaagt acgacaagct caaactgggc aaggccatcg acaaaaacgc
     4321 caagtttatc gctagcttct acaagaatga tctgatcaag ctggacggcg agatctacaa
     4381 gatcatcggc gtgaatagcg acaccagaaa catgatcgaa ctggatctgc ctgacatcag
     4441 atacaaagaa tactgcgagc tgaacaatat caagggcgaa cctcacatca aaaagaccat
     4501 cggcaaaaag gtgaatagca tcgaaaaact gacaaccgac gtgctgggca acgtgttcac
     4561 caacacccag tacacaaaac ctcagctgct gttcaagcga ggaaatgact acaaggacga
     4621 cgacgacaag gactataagg acgatgatga taaggattac aaagatgacg acgataagcc
     4681 ccccaagaag aagaggaagg tggaggccag ctaagaattc aataaaagat ctttattttc
     4741 attagatctg tgtgttggtt ttttgtgtgt aagaattccg gccgcaggaa cccctagtga
     4801 tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg
     4861 tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc
     4921 tgcaggatcg gatcccgggc ccgtcgactg cagaggcctg catgcaagct tggcgtaatc
     4981 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     5041 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     5101 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     5161 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     5221 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     5281 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5341 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5401 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5461 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     5521 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5581 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5641 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5701 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5761 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5821 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5881 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     5941 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     6001 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     6061 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     6121 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     6181 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6241 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     6301 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6361 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6421 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     6481 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6541 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6601 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6661 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6721 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6781 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6841 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     6901 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6961 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     7021 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     7081 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     7141 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     7201 tcgtc
//
