LOCUS       sequence-417988-        7291 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Coding for CjSp D8A alongside Bilbo sgRNA targeting CAG repeats.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7291)
  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 7291)
  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 Bilbo -eCMV-CjSpD8A-3XFLA...
FEATURES             Location/Qualifiers
     source          1..7291
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     100..119
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     313..335
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     327..344
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     328..344
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   381..521
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   392..521
                     /label=AAV2 ITR
     promoter        554..644
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     565..584
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     enhancer        838..1141
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1142..1345
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1296..1316
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1384..1393
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2695..2706)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             4693..4764
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     CDS             4768..4788
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4807..4855
                     /note="synthetic polyadenylation signal"
     repeat_region   4872..5012
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4872..5001
                     /label=AAV2 ITR
     primer_bind     complement(5070..5086)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5070..5086)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5083..5105)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5094..5110
                     /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(5118..5124,5125..5142,5143..5148))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5163..5184
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5472..6060)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5552..5571)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6231..7022,7023..7091))
                     /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     6854..6873
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7092..7196)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7264..7282
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
       61 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac
      121 tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca
      181 tcaggcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
      241 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa
      301 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattc gagctcggta
      361 cctcgcgaat gcatctagat cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc
      421 ccgggcaaag cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc
      481 gcgcagagag ggagtggcca actccatcac taggggttcc tgcggccccc cctaggtagg
      541 ctaggctagg cgtatatttg catgtcgcta tgtgttctgg gaaatcacca taaacgtgaa
      601 atgtctttgg atttgggaat cttataagtt ctgtatgaga ccactctttc ccgctgctgc
      661 tgctgctgct ggttttagtc cctgaaaagg gactaaaatg gctagtccgt tatcaacttg
      721 aaaaagtggc accgagtcgg tgcttttttt tctagagggt accggggccc gcctaggtag
      781 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
      841 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
      901 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
      961 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
     1021 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
     1081 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
     1141 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
     1201 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
     1261 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
     1321 gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct agcgctaccg
     1381 gtcgccacca tggcccgcat cctggccttt gccattggca tctcttcaat cggctgggcc
     1441 ttctcagaga atgacgagct gaaggactgc ggagtgcgga tcttcaccaa ggtcgagaac
     1501 cccaagaccg gcgagtctct ggccctgccg cggcgactgg cgaggtctgc tagaaagcgc
     1561 ctggccagac gaaaggccag actcaaccac ctgaagcacc tgatcgccaa cgaattcaaa
     1621 ctcaactacg aggactacca gagcttcgac gagtccctgg ctaaggcgta caagggatct
     1681 ctgatctccc cttacgagtt gagatttagg gctctgaatg agctcctctc taaacaggac
     1741 ttcgcccgcg tcatcctgca tattgccaaa cggagggggt acgacgacat taaaaactcc
     1801 gacgacaagg agaagggcgc aatcctcaag gccatcaagc agaacgagga gaagctcgcc
     1861 aactaccaga gcgtggggga gtacctgtac aaggagtact ttcagaaatt caaggagaac
     1921 agcaaggagt tcaccaatgt gagaaacaag aaggagtcct acgaaagatg cattgcgcag
     1981 agctttctta aggacgagct caaactgatc ttcaagaagc agagagagtt cggcttctct
     2041 ttcagcaaaa agttcgaaga ggaggtgctg agcgtggcct tctacaagcg agcgctgaag
     2101 gacttcagcc acctggtggg taattgttcc ttcttcaccg acgagaagag agcccccaaa
     2161 aatagcccac tggcctttat gttcgttgcc cttaccagaa tcatcaacct gctcaacaat
     2221 ctgaagaaca cagagggaat tctctataca aaggacgacc tcaacgccct gcttaacgag
     2281 gtgctcaaga acgggactct gacatacaag caaacaaaaa agctcctggg tctgtctgac
     2341 gactacgagt ttaagggaga aaaaggcacc tacttcatcg agttcaagaa gtacaaggaa
     2401 ttcatcaagg ccctcggcga gcacaatctc agccaggacg acctgaacga gatcgccaag
     2461 gacatcacac tgatcaagga cgaaattaaa ctcaagaaag ccctggccaa gtacgacctc
     2521 aaccaaaacc agattgactc cctctctaag ttggagttca aggaccacct gaacatcagc
     2581 ttcaaggctc tgaagctggt gacacccctg atgctggaag gtaagaagta cgacgaagct
     2641 tgtaacgagc tgaatctgaa ggtcgccatc aacgaggaca agaaggactt cctcccggcc
     2701 ttcaatgaga cctactacaa ggacgaagtc acaaaccctg tcgtcttgag agccatcaag
     2761 gagtacagaa aggtgctcaa cgccctgctg aagaagtacg gaaaggtaca caaaatcaat
     2821 attgaacttg ctagagaagt tggcaaaaac cacagccagc gggccaagat cgaaaaggag
     2881 cagaacgaga actacaaggc caagaaagat gccgagctcg aatgcgagaa gcttggcctg
     2941 aagatcaact caaagaacat cttgaagctc agacttttca aagagcagaa agaattctgt
     3001 gcgtattctg gcgagaagat taaaatctca gatcttcaag acgagaagat gcttgaaatc
     3061 gaccacatct acccttactc aaggtctttc gacgactcat acatgaacaa agtcctggtg
     3121 ttcaccaagc agaaccagga gaaactgaac cagacaccat tcgaggcctt cggcaacgac
     3181 agcgcgaagt ggcagaagat cgaggtcctc gccaaaaacc tccccacgaa aaagcaaaag
     3241 cgaatcttgg acaagaacta caaggacaag gagcagaaga actttaagga tagaaacttg
     3301 aacgacactc ggtatatcgc taggctcgtc ttgaattata ccaaggacta cttggatttc
     3361 ctccccctga gcgacgatga aaacaccaag ctcaacgata cccagaaagg ctccaaagtg
     3421 cacgtcgagg caaagtcagg tatgctcact tccgccctga ggcacacctg gggattcagc
     3481 gcgaaggata ggaacaacca cctgcaccac gccattgacg ctgtgattat tgcctatgca
     3541 aacaactcca ttgttaaggc tttcagcgat ttcaagaaag agcaggagtc caattcagct
     3601 gagttgtatg cgaagaagat cagcgaactg gattacaaga acaagagaaa gttcttcgaa
     3661 cccttctccg gcttcaggca gaaggtcctc gataaaattg atgagatctt cgtctccaag
     3721 cccgagagga aaaagcccag cggtgcgctt catgaggaga cattcaggaa ggaggaggag
     3781 ttctaccaga gctacggcgg aaaggagggc gtactgaagg cccttgaatt gggaaagatc
     3841 agaaaagtca acggcaaaat cgttaagaat ggcggcggcg gcagcggcgg caccggagga
     3901 ttcagtaagg aaagcatact gccgaaaagg aattccgaca agctgatcgc taggaagaag
     3961 gactgggatc ccaaaaaata cgggggattc gactccccca cagttgcgta cagcgtgctc
     4021 gtggtggcaa aagtggagaa ggggaaatcc aaaaaactaa aatcagtcaa agagctgctt
     4081 ggcattacaa tcatggagag gtcttccttc gagaagaatc caattgactt cctcgaggct
     4141 aagggataca aggaggtgaa gaaggacttg ataatcaaac tgccgaagta cagcttgttc
     4201 gagctcgaaa atggccgcaa gaggatgctg gctagcgctg gcgagctcca gaagggcaac
     4261 gagctggccc tgcccagcaa gtacgtgaac ttcctttatc tcgcatcaca ctacgagaag
     4321 ctcaagggat cacccgagga caacgagcag aagcagctgt ttgtggagca gcacaagcac
     4381 tatctcgatg aaattattga acagatctcc gaattttcta aacgggtaat tctcgcagac
     4441 gctaacctcg acaaagtgct gagcgcatac aataaacatc gggacaagcc aattagggag
     4501 caggctgaga acatcatcca tcttttcacc ttgacaaatc tcggcgctcc agccgccttc
     4561 aagtacttcg acaccaccat cgataggaag cgatacacat caacgaaaga ggtgctggac
     4621 gccactctga tccaccagag catcaccggc ctgtacgaaa cgagaatcga cttgtctcag
     4681 ctgggcggag atgactacaa ggacgacgac gacaaggact ataaggacga tgatgataag
     4741 gattacaaag atgacgacga taagcccccc aagaagaaga ggaaggtgga ggccagctaa
     4801 gaattcaata aaagatcttt attttcatta gatctgtgtg ttggtttttt gtgtgtaaga
     4861 attccggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     4921 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc
     4981 agtgagcgag cgagcgcgca gctgcctgca ggatcggatc ccgggcccgt cgactgcaga
     5041 ggcctgcatg caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     5101 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     5161 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     5221 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5281 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5341 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5401 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5461 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5521 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5581 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5641 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     5701 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5761 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5821 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5881 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     5941 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     6001 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     6061 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     6121 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     6181 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     6241 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     6301 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     6361 atgataccgc gagatccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     6421 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6481 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6541 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6601 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6661 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6721 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6781 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6841 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6901 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     6961 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     7021 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     7081 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     7141 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     7201 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     7261 aaaaataggc gtatcacgag gccctttcgt c
//
