LOCUS       sequence-420081-        7572 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Coding for SaSp D10A Cas9 alongside Merry sgRNA targeting CAG 
            repeats.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7572)
  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 7572)
  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 Merry-eCMV-SaSpD10A+linke...
FEATURES             Location/Qualifiers
     source          1..7572
                     /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        879..1182
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1183..1386
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1337..1357
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     RBS             3064..3072
                     /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             4974..5045
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     CDS             5049..5069
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    5088..5136
                     /note="synthetic polyadenylation signal"
     repeat_region   5153..5293
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5153..5282
                     /label=AAV2 ITR
     primer_bind     complement(5351..5367)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5351..5367)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5364..5386)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5375..5391
                     /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(5399..5405,5406..5423,5424..5429))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5444..5465
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5753..6341)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5833..5852)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6512..7303,7304..7372))
                     /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     7135..7154
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7373..7477)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7545..7563
                     /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 taaaacaagg caaaatgccg aacttgaaaa
      781 agtgtttttt ttctagaggg taccggggcc cgcctaggta gttattaata gtaatcaatt
      841 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
      901 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
      961 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     1021 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     1081 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
     1141 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
     1201 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
     1261 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
     1321 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
     1381 agagctggtt tagtgaaccg tcagatccgc tagcgctacc ggtcgccacc atgaagagga
     1441 attacattct gggcctggcc atcgggatca cctctgtggg gtatggcatt atcgactacg
     1501 agacaagaga cgtgatcgac gcaggcgtca gactcttcaa ggaggccaac gtggagaata
     1561 acgaaggcag gcggagcaag aggggtgcaa ggagattgaa aagaaggcgg cgccacagaa
     1621 tccagagggt gaagaagctc cttttcgact acaacttgct caccgatcat agcgagctgt
     1681 ccggcatcaa tccttacgag gctagggtca agggattgag ccagaagttg tcagaggagg
     1741 agttcagcgc agcactgctc catcttgcca agaggagagg agtccacaac gtgaatgagg
     1801 tggaggaaga tactgggaat gagctgagca cgaaggagca gatctctaga aactcaaaag
     1861 ccttggaaga aaagtatgtt gctgaactcc agcttgaaag actcaagaag gacggggagg
     1921 tgaggggctc aattaacagg ttcaagacat ctgactatgt gaaagaagca aagcagctcc
     1981 tcaaagtgca aaaagcttac caccagctgg accaaagttt tatcgacacg tacatcgatc
     2041 ttttggaaac acgacgaacg tattacgaag gaccaggtga aggctcaccc tttggctgga
     2101 aagacattaa agagtggtac gaaatgttga tgggacactg tacatacttc cctgaagagc
     2161 tgcgcagcgt caagtacgcc tataacgccg acctgtacaa cgcccttaac gacctcaaca
     2221 atttggtgat cactagagac gagaacgaaa agctcgagta ctacgaaaaa ttccaaatta
     2281 tcgaaaatgt cttcaagcag aagaaaaagc ctacactcaa acagattgcc aaagagatat
     2341 tggtaaacga agaggatatc aaagggtaca gagtcacttc caccgggaag ccggagttca
     2401 ctaatctcaa ggtttatcac gacataaagg acataaccgc ccggaaagag atcattgaga
     2461 acgccgagct tctggaccag attgcaaaaa ttcttactat ctaccaatca tccgaggaca
     2521 tccaggagga gctcaccaat cttaatagcg agctcacgca agaagaaatt gagcaaatta
     2581 gcaatctcaa ggggtacacc ggaacccata atttgagcct taaggctatt aacttgattc
     2641 tggacgagct ttggcacacg aacgataatc agattgccat ttttaaccgg cttaaactcg
     2701 ttccgaagaa agttgatttg agtcaacaaa aggaaatacc taccactttg gtagacgatt
     2761 ttattctctc ccctgtggta aagcgatcct ttatccagtc aatcaaagtg attaatgcaa
     2821 tcattaaaaa gtacgggctg cccaatgaca ttattatcga gctcgcaagg gagaagaaca
     2881 gcaaagatgc ccagaagatg ataaacgaga tgcagaagcg gaatcgacag acaaatgaaa
     2941 ggattgaaga aataatccga acaacaggga aggaaaatgc taagtatctt atcgaaaaga
     3001 ttaagctcca cgatatgcag gagggcaagt gcctttactc tctcgaggcg attccattgg
     3061 aggacctcct taacaatccg ttcaactacg aggtggatca catcatcccc aggagcgtga
     3121 gcttcgacaa ctcattcaac aacaaggtgt tagtgaagca ggaggagaac agcaagaaag
     3181 gtaataggac cccattccag taccttagta gctctgactc caagatcagc tacgagactt
     3241 tcaagaagca catactgaac ttagccaagg gtaaaggccg cattagcaaa accaagaagg
     3301 agtatttgct ggaagaaagg gacatcaacc ggttctcagt ccagaaggac ttcatcaaca
     3361 ggaacctggt ggacacaaga tacgctacca ggggcctgat gaacctcttg agatcttact
     3421 tccgggtcaa taacctggat gttaaggtca agtctatcaa cggcggcttc accagcttcc
     3481 tgagaagaaa gtggaagttc aagaaggagc gaaacaaggg gtacaaacac catgccgagg
     3541 acgctctgat aatcgctaac gccgacttca tcttcaagga gtggaagaag ctggacaaag
     3601 ccaagaaggt gatggaaaac cagatgttcg aagagaagca ggctgagtcc atgccagaga
     3661 tcgagacaga gcaggaatac aaggaaatct tcatcacccc tcaccagatc aaacatatta
     3721 aggatttcaa agactataag tactcccata gagttgacaa aaagcccaac agagaactga
     3781 tcaacgacac tctctactct accagaaagg acgataaagg taacactctt attgtcaaca
     3841 atctcaatgg cctgtacgac aaggacaacg ataagctgaa gaaactgatc aacaaatcac
     3901 ccgagaagct cctgatgtac caccacgacc cacagacata ccagaagttg aagctcatca
     3961 tggaacagta cggcgacgag aagaaccccc tgtacaaata ctacgaggaa acggggaatt
     4021 acctcaccaa gtactctaaa aaggacaacg gacccgttat taaaaagatc aaatattatg
     4081 gtaacaagct taatgctcat ctggacatca ccgatgatta ccccaattcc agaaataagg
     4141 tggtcaaact gtctggcggc ggcagcggcg gcaccggagg attcagtaag gaaagcatac
     4201 tgccgaaaag gaattccgac aagctgatcg ctaggaagaa ggactgggat cccaaaaaat
     4261 acgggggatt cgactccccc acagttgcgt acagcgtgct cgtggtggca aaagtggaga
     4321 aggggaaatc caaaaaacta aaatcagtca aagagctgct tggcattaca atcatggaga
     4381 ggtcttcctt cgagaagaat ccaattgact tcctcgaggc taagggatac aaggaggtga
     4441 agaaggactt gataatcaaa ctgccgaagt acagcttgtt cgagctcgaa aatggccgca
     4501 agaggatgct ggctagcgct ggcgagctcc agaagggcaa cgagctggcc ctgcccagca
     4561 agtacgtgaa cttcctttat ctcgcatcac actacgagaa gctcaaggga tcacccgagg
     4621 acaacgagca gaagcagctg tttgtggagc agcacaagca ctatctcgat gaaattattg
     4681 aacagatctc cgaattttct aaacgggtaa ttctcgcaga cgctaacctc gacaaagtgc
     4741 tgagcgcata caataaacat cgggacaagc caattaggga gcaggctgag aacatcatcc
     4801 atcttttcac cttgacaaat ctcggcgctc cagccgcctt caagtacttc gacaccacca
     4861 tcgataggaa gcgatacaca tcaacgaaag aggtgctgga cgccactctg atccaccaga
     4921 gcatcaccgg cctgtacgaa acgagaatcg acttgtctca gctgggcgga gatgactaca
     4981 aggacgacga cgacaaggac tataaggacg atgatgataa ggattacaaa gatgacgacg
     5041 ataagccccc caagaagaag aggaaggtgg aggccagcta agaattcaat aaaagatctt
     5101 tattttcatt agatctgtgt gttggttttt tgtgtgtaag aattccggcc gcaggaaccc
     5161 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     5221 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     5281 agctgcctgc aggatcggat cccgggcccg tcgactgcag aggcctgcat gcaagcttgg
     5341 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     5401 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     5461 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5521 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     5581 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     5641 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5701 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5761 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5821 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5881 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5941 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     6001 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     6061 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     6121 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6181 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6241 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6301 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6361 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6421 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     6481 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     6541 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     6601 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6661 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     6721 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     6781 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     6841 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     6901 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6961 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     7021 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     7081 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     7141 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     7201 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     7261 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     7321 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     7381 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     7441 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     7501 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     7561 ggccctttcg tc
//
