LOCUS       sequence-449594-        7206 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid codes for the Slug-KH nickase with a sg(CTG)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-sgCTG-SlugD10A-3XFLAG-SV40 NLS-ITR2
FEATURES             Location/Qualifiers
     source          1..7206
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1305..1508)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1334..1354)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1509..1812
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_RNA        complement(1908..1983)
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     promoter        complement(2011..2101)
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     complement(2071..2090)
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     repeat_region   2134..2274
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2134..2263
                     /label=AAV2 ITR
     primer_bind     complement(2311..2328)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2311..2327)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2320..2342)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2536..2555)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2655..2677
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2715..2733)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2801..2905
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2906..2974,2975..3766)
                     /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(3124..3143)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3937..4525
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4426..4445
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    4813..4834
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4849..4854,4855..4872,4873..4879)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4887..4903
                     /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     4892..4914
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4911..4927
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4911..4927
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4985..5125
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4996..5125
                     /label=AAV2 ITR
     polyA_signal    5142..5190
                     /note="synthetic polyadenylation signal"
     CDS             complement(5209..5229)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(5233..5304)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
ORIGIN
        1 gaagatttcc atctgggccc ggctagaata ccactgctct tccaggacca gccggatgca
       61 tttcaggctg agccggtggg tcccggtgta gccagtcagc tgggcgatgt tttccttatc
      121 ttcctcgttc agcaggatgt ccagttccgt cagcttagat ttgatgctat ccttgtcctg
      181 gtagatggtc aggatctcag cgatctggtc cagcacgtct tcgttctcca ggatgctctg
      241 gtcaaacagc acgcttttca ggtcgtggta gagcttaaac tcggtaaact ggggcttgcc
      301 agactttgtg attctgtagc ccttgatatc ttcagggttg acattgatct cattggcgat
      361 ctgcttcagt gtaggtttct tcttctgctt gaacacgttc tcgatgatgt gatacttctc
      421 atgatactcg agcttagaca gtccatctct ttggatgacc aggttgttca ggtcgttcag
      481 agcgttgaac aggtcggcac tgtaggcgta tttcacgctg gccagctcat ctgggaagta
      541 ggtgcagtgg cccatcagtg tctcgtacca agccttaggg tcgccttccc agccatatgg
      601 actgcccttg ccggggccct cgaagtattc ccgtctcatc tcaaccagtt cgatgtactt
      661 gttgatgaag ttctcgtcca gctggtggaa gttcttctgc acattcagca gctggatgat
      721 ttccttgatg atatcagcgg tcttgaaccg gtttttctct ccccgcacct ggccctcatt
      781 cattctttcc agctggatct ggcacacaaa tttgtctttc agcagcttgc tgttcttatt
      841 cagctgttcc ttggtgctga gctcgttccc cacgtcgtcg ttgctgtcga tcacgtcgat
      901 cttgtggatg cctcttctct tggcgatatg gagcagggca atcacgagtt cgtccttgct
      961 cagggcttca gacaggccct tcacgcggat ggcgtagggg tttgtagact gaggaatctg
     1021 ggactgatcc agcagattat aatcctcgag cagcttcttc acccgttcaa gtctgtgaat
     1081 gcggcgtcgc ttcagtcttc tgcttccacg tttggacctt cttccctcgt tgttttccac
     1141 gttggcctcg gggaacagtc taacgccggc atcgatgata ttctttgtct cgtagtcgat
     1201 caggccgtag cccacgctgg tgattccgat ggccagtccc agtatgaatt tttggttcat
     1261 ggtggcgacc ggtagcgcta gcggatctga cggttcacta aaccagctct gcttatatag
     1321 acctcccacc gtacacgcct accgcccatt tgcgtcaatg gggcggagtt gttacgacat
     1381 tttggaaagt cccgttgatt ttggtgccaa aacaaactcc cattgacgtc aatggggtgg
     1441 agacttggaa atccccgtga gtcaaaccgc tatccacgcc cattgatgta ctgccaaaac
     1501 cgcatcacca tggtaatagc gatgactaat acgtagatgt actgccaagt aggaaagtcc
     1561 cataaggtca tgtactgggc ataatgccag gcgggccatt taccgtcatt gacgtcaata
     1621 gggggcgtac ttggcatatg atacacttga tgtactgcca agtgggcagt ttaccgtaaa
     1681 tactccaccc attgacgtca atggaaagtc cctattggcg ttactatggg aacatacgtc
     1741 attattgacg tcaatgggcg ggggtcgttg ggcggtcagc caggcgggcc atttaccgta
     1801 agttatgtaa cgcggaactc catatatggg ctatgaacta atgaccccgt aattgattac
     1861 tattaataac tacctaggcg ggccccggta ccctctagaa aaaaaaatct cgccaacaag
     1921 ttgacgagat aaacacggca ttttgccttg ttttagtaga ttctgtttcc agagtactaa
     1981 aaccagcagc agcagcagca gcgggaaaga gtggtctcat acagaactta taagattccc
     2041 aaatccaaag acatttcacg tttatggtga tttcccagaa cacatagcga catgcaaata
     2101 tacgcctagc ctagcctacc tagggggggc cgcaggaacc cctagtgatg gagttggcca
     2161 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     2221 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggatctag
     2281 atgcattcgc gaggtaccga gctcgaattc actggccgtc gttttacaac gtcgtgactg
     2341 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     2401 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     2461 cgaatggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     2521 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     2581 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     2641 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     2701 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     2761 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     2821 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     2881 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     2941 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     3001 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     3061 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     3121 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     3181 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     3241 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     3301 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     3361 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     3421 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     3481 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     3541 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     3601 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     3661 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     3721 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     3781 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     3841 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     3901 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     3961 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     4021 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     4081 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     4141 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     4201 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     4261 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     4321 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     4381 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     4441 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     4501 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     4561 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     4621 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     4681 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
     4741 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     4801 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
     4861 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
     4921 ctatgaccat gattacgcca agcttgcatg caggcctctg cagtcgacgg gcccgggatc
     4981 cgatcctgca ggcagctgcg cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg
     5041 cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg
     5101 gccaactcca tcactagggg ttcctgcggc cggaattctt acacacaaaa aaccaacaca
     5161 cagatctaat gaaaataaag atcttttatt gaattcttag ctggcctcca ccttcctctt
     5221 cttcttgggg ggcttatcgt cgtcatcttt gtaatcctta tcatcatcgt ccttatagtc
     5281 cttgtcgtcg tcgtccttgt agtcatttcc tcgcttgaac agcagctgag gttttgtgta
     5341 ctgggtgttg gtgaacacgt tgcccagcac gtcggttgtc agtttttcga tgctattcac
     5401 ctttttgccg atggtctttt tgatgtgagg ttcgcccttg atattgttca gctcgcagta
     5461 ttctttgtat ctgatgtcag gcagatccag ttcgatcatg tttctggtgt cgctattcac
     5521 gccgatgatc ttgtagatct cgccgtccag cttgatcaga tcattcttgt agaagctagc
     5581 gataaacttg gcgtttttgt cgatggcctt gcccagtttg agcttgtcgt acttctgttc
     5641 gggtatgtag tagtaattgt ctttcttcag cacgtccagg tagctgatgg tgatgaactt
     5701 atatcccttg tcggtcaggt agacgtcgaa tctataaggc ttgatgctca gcttcaccaa
     5761 cttcttggtg ctgctcttga actggtgggt cacatctagg tggctgccca gcttgtttcc
     5821 aatgtacttc aggcttttca cgatagggcc gttgtttttt ttgctgtatt tggtcaggta
     5881 ctcgcctgtt tcctcgtggt acttggccag aggattcttc tcattagcgt actgcttcat
     5941 gatcacttcc aacttctcga aggtccgcgg atcgtgttgg tacatgagga acttctcggg
     6001 tgacttgtca aactgcttct tcagggtggt attatctttg gcatagatat ctttgatggt
     6061 ctgcacgatg taggtggagt tgtccttctt ccgggtgctg tacagtgtat cgttgatcag
     6121 ctttctgtta ggcttcttgt ccactctgtg gctgtacttg aagtttctga agtccttgat
     6181 gtcctggacc tgcttaggga tgatgaacat ttcgctgtag ttatcctcgc tatccacctg
     6241 gatatccagc tgcttgctct cgatctcagg tttttccagc acagagttca cggctttcag
     6301 cttcttgttc tctttgaaca ggaaatcggc attagcgata atcagggcgt cctcggcgtg
     6361 gtgcttgtag ccgtggttcc gttccttctt aaacttccac actttccgca ggtaatcggt
     6421 gaagctgccg ttgatcgttt tcaccttcac gttcatattg ttggcgctga agtaggcctt
     6481 caggtagttt gtcagctcgg ctgtggcgta ccgggtatcc accaggtttc tattgatgaa
     6541 ctctttttgc acctcgaact tgttgatgtc ccgttcttcc agcagatact ctttcttttt
     6601 tttgctgatt ctatcctggg acttgctcag gttcaggatg tgttgtttaa actggttgta
     6661 agagagcttg gacttgccgc tgttaaagta ctggtaaggg gtcaggttgg attttttgga
     6721 gttctcgctc tgcttcacga gcactttgtt gtggtaagag ttgtcgaagg acacagatct
     6781 tggaatgatg tggtccacct cgtagtggtt ggggttgttc agcaggtcct ccagagggat
     6841 gctttccagt gagtacaggc acttgccctc ctgctcgtcg tgcagtctaa tcttctcgac
     6901 cagtctcttg gcgttctggt tgccatactt gccgatgatt tcgttaattc tctttctggt
     6961 gttctcgttc tttttctgca tctcattgat gaacttctgc ttgtccttgc tattattttc
     7021 tctggccagc tcgatgatga tatcttcagg cacgccgtac ttttctatga tcttgttaat
     7081 aaggttaatg gcctggccga aggtcctctt caccacaggg ctcaggatga actcgtcgat
     7141 catagccttc gggatcttgt tggcggctgt caggttgatc ttcttaggct taatgttcag
     7201 gtgggc
//
