LOCUS       sequence-464595-        7567 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Coding for SaSp D10A Cas9 alongside Frodo sgRNA targeting CAG 
            repeats.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7567)
  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 7567)
  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 Frodo-eCMV-SaSpD10A (Neur...
FEATURES             Location/Qualifiers
     source          1..7567
                     /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"
     enhancer        892..1195
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1196..1399
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1350..1370
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     RBS             3077..3085
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             4969..5040
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     CDS             5044..5064
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    5083..5131
                     /note="synthetic polyadenylation signal"
     repeat_region   5148..5288
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5148..5277
                     /label=AAV2 ITR
     primer_bind     complement(5346..5362)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5346..5362)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5359..5381)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5370..5386
                     /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(5394..5400,5401..5418,5419..5424))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5439..5460
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5748..6336)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5828..5847)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6507..7298,7299..7367))
                     /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     7130..7149
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7368..7472)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7540..7558
                     /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 cccgctgctg ctgctgctgc
      721 tggttttagt actctggaaa cagaatctac taaaacggct agttatcaac ttgaaaaagt
      781 ggcaccgagt cggtgcattt tttttctaga gggtaccggg gcccgcctag gtagttatta
      841 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
      901 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
      961 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
     1021 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
     1081 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
     1141 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
     1201 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
     1261 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
     1321 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
     1381 ggtctatata agcagagctg gtttagtgaa ccgtcagatc cgctagcgct accggtcgcc
     1441 accatgaaga ggaattacat tctgggcctg gccatcggga tcacctctgt ggggtatggc
     1501 attatcgact acgagacaag agacgtgatc gacgcaggcg tcagactctt caaggaggcc
     1561 aacgtggaga ataacgaagg caggcggagc aagaggggtg caaggagatt gaaaagaagg
     1621 cggcgccaca gaatccagag ggtgaagaag ctccttttcg actacaactt gctcaccgat
     1681 catagcgagc tgtccggcat caatccttac gaggctaggg tcaagggatt gagccagaag
     1741 ttgtcagagg aggagttcag cgcagcactg ctccatcttg ccaagaggag aggagtccac
     1801 aacgtgaatg aggtggagga agatactggg aatgagctga gcacgaagga gcagatctct
     1861 agaaactcaa aagccttgga agaaaagtat gttgctgaac tccagcttga aagactcaag
     1921 aaggacgggg aggtgagggg ctcaattaac aggttcaaga catctgacta tgtgaaagaa
     1981 gcaaagcagc tcctcaaagt gcaaaaagct taccaccagc tggaccaaag ttttatcgac
     2041 acgtacatcg atcttttgga aacacgacga acgtattacg aaggaccagg tgaaggctca
     2101 ccctttggct ggaaagacat taaagagtgg tacgaaatgt tgatgggaca ctgtacatac
     2161 ttccctgaag agctgcgcag cgtcaagtac gcctataacg ccgacctgta caacgccctt
     2221 aacgacctca acaatttggt gatcactaga gacgagaacg aaaagctcga gtactacgaa
     2281 aaattccaaa ttatcgaaaa tgtcttcaag cagaagaaaa agcctacact caaacagatt
     2341 gccaaagaga tattggtaaa cgaagaggat atcaaagggt acagagtcac ttccaccggg
     2401 aagccggagt tcactaatct caaggtttat cacgacataa aggacataac cgcccggaaa
     2461 gagatcattg agaacgccga gcttctggac cagattgcaa aaattcttac tatctaccaa
     2521 tcatccgagg acatccagga ggagctcacc aatcttaata gcgagctcac gcaagaagaa
     2581 attgagcaaa ttagcaatct caaggggtac accggaaccc ataatttgag ccttaaggct
     2641 attaacttga ttctggacga gctttggcac acgaacgata atcagattgc catttttaac
     2701 cggcttaaac tcgttccgaa gaaagttgat ttgagtcaac aaaaggaaat acctaccact
     2761 ttggtagacg attttattct ctcccctgtg gtaaagcgat cctttatcca gtcaatcaaa
     2821 gtgattaatg caatcattaa aaagtacggg ctgcccaatg acattattat cgagctcgca
     2881 agggagaaga acagcaaaga tgcccagaag atgataaacg agatgcagaa gcggaatcga
     2941 cagacaaatg aaaggattga agaaataatc cgaacaacag ggaaggaaaa tgctaagtat
     3001 cttatcgaaa agattaagct ccacgatatg caggagggca agtgccttta ctctctcgag
     3061 gcgattccat tggaggacct ccttaacaat ccgttcaact acgaggtgga tcacatcatc
     3121 cccaggagcg tgagcttcga caactcattc aacaacaagg tgttagtgaa gcaggaggag
     3181 aacagcaaga aaggtaatag gaccccattc cagtacctta gtagctctga ctccaagatc
     3241 agctacgaga ctttcaagaa gcacatactg aacttagcca agggtaaagg ccgcattagc
     3301 aaaaccaaga aggagtattt gctggaagaa agggacatca accggttctc agtccagaag
     3361 gacttcatca acaggaacct ggtggacaca agatacgcta ccaggggcct gatgaacctc
     3421 ttgagatctt acttccgggt caataacctg gatgttaagg tcaagtctat caacggcggc
     3481 ttcaccagct tcctgagaag aaagtggaag ttcaagaagg agcgaaacaa ggggtacaaa
     3541 caccatgccg aggacgctct gataatcgct aacgccgact tcatcttcaa ggagtggaag
     3601 aagctggaca aagccaagaa ggtgatggaa aaccagatgt tcgaagagaa gcaggctgag
     3661 tccatgccag agatcgagac agagcaggaa tacaaggaaa tcttcatcac ccctcaccag
     3721 atcaaacata ttaaggattt caaagactat aagtactccc atagagttga caaaaagccc
     3781 aacagagaac tgatcaacga cactctctac tctaccagaa aggacgataa aggtaacact
     3841 cttattgtca acaatctcaa tggcctgtac gacaaggaca acgataagct gaagaaactg
     3901 atcaacaaat cacccgagaa gctcctgatg taccaccacg acccacagac ataccagaag
     3961 ttgaagctca tcatggaaca gtacggcgac gagaagaacc ccctgtacaa atactacgag
     4021 gaaacgggga attacctcac caagtactct aaaaaggaca acggacccgt tattaaaaag
     4081 atcaaatatt atggtaacaa gcttaatgct catctggaca tcaccgatga ttaccccaat
     4141 tccagaaata aggtggtcaa actgtctacc ggaggattca gtaaggaaag catactgccg
     4201 aaaaggaatt ccgacaagct gatcgctagg aagaaggact gggatcccaa aaaatacggg
     4261 ggattcgact cccccacagt tgcgtacagc gtgctcgtgg tggcaaaagt ggagaagggg
     4321 aaatccaaaa aactaaaatc agtcaaagag ctgcttggca ttacaatcat ggagaggtct
     4381 tccttcgaga agaatccaat tgacttcctc gaggctaagg gatacaagga ggtgaagaag
     4441 gacttgataa tcaaactgcc gaagtacagc ttgttcgagc tcgaaaatgg ccgcaagagg
     4501 atgctggcta gcgctggcga gctccagaag ggcaacgagc tggccctgcc cagcaagtac
     4561 gtgaacttcc tttatctcgc atcacactac gagaagctca agggatcacc cgaggacaac
     4621 gagcagaagc agctgtttgt ggagcagcac aagcactatc tcgatgaaat tattgaacag
     4681 atctccgaat tttctaaacg ggtaattctc gcagacgcta acctcgacaa agtgctgagc
     4741 gcatacaata aacatcggga caagccaatt agggagcagg ctgagaacat catccatctt
     4801 ttcaccttga caaatctcgg cgctccagcc gccttcaagt acttcgacac caccatcgat
     4861 aggaagcgat acacatcaac gaaagaggtg ctggacgcca ctctgatcca ccagagcatc
     4921 accggcctgt acgaaacgag aatcgacttg tctcagctgg gcggagatga ctacaaggac
     4981 gacgacgaca aggactataa ggacgatgat gataaggatt acaaagatga cgacgataag
     5041 ccccccaaga agaagaggaa ggtggaggcc agctaagaat tcaataaaag atctttattt
     5101 tcattagatc tgtgtgttgg ttttttgtgt gtaagaattc cggccgcagg aacccctagt
     5161 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     5221 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     5281 cctgcaggat cggatcccgg gcccgtcgac tgcagaggcc tgcatgcaag cttggcgtaa
     5341 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     5401 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     5461 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     5521 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     5581 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     5641 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     5701 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     5761 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     5821 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     5881 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     5941 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     6001 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     6061 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     6121 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     6181 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     6241 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     6301 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     6361 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     6421 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     6481 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     6541 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     6601 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     6661 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     6721 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     6781 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     6841 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     6901 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     6961 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     7021 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     7081 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     7141 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     7201 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     7261 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     7321 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     7381 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     7441 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     7501 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     7561 tttcgtc
//
