LOCUS       sequence-420033-        7555 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Coding for SaSp Cas9 alongside Merry sgRNA targeting GFP Target 4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7555)
  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 7555)
  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-GFP sgRNA Merry Target 4- eCMV-
            ...
FEATURES             Location/Qualifiers
     source          1..7555
                     /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        880..1183
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1184..1387
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1338..1358
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     RBS             3065..3073
                     /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             4957..5028
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     CDS             5032..5052
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    5071..5119
                     /note="synthetic polyadenylation signal"
     repeat_region   5136..5276
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5136..5265
                     /label=AAV2 ITR
     primer_bind     complement(5334..5350)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5334..5350)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5347..5369)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5358..5374
                     /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(5382..5388,5389..5406,5407..5412))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5427..5448
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5736..6324)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5816..5835)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6495..7286,7287..7355))
                     /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     7118..7137
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7356..7460)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7528..7546
                     /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 cccgggcgag gagctgttca
      721 ccggttttag tactctggaa acagaatcta ctaaaacaag gcaaaatgcc gaacttgaaa
      781 aagtgttttt tttctagagg gtaccggggc ccgcctaggt agttattaat agtaatcaat
      841 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
      901 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
      961 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     1021 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
     1081 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
     1141 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
     1201 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
     1261 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
     1321 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
     1381 cagagctggt ttagtgaacc gtcagatccg ctagcgctac cggtcgccac catgaagagg
     1441 aattacattc tgggcctgga catcgggatc acctctgtgg ggtatggcat tatcgactac
     1501 gagacaagag acgtgatcga cgcaggcgtc agactcttca aggaggccaa cgtggagaat
     1561 aacgaaggca ggcggagcaa gaggggtgca aggagattga aaagaaggcg gcgccacaga
     1621 atccagaggg tgaagaagct ccttttcgac tacaacttgc tcaccgatca tagcgagctg
     1681 tccggcatca atccttacga ggctagggtc aagggattga gccagaagtt gtcagaggag
     1741 gagttcagcg cagcactgct ccatcttgcc aagaggagag gagtccacaa cgtgaatgag
     1801 gtggaggaag atactgggaa tgagctgagc acgaaggagc agatctctag aaactcaaaa
     1861 gccttggaag aaaagtatgt tgctgaactc cagcttgaaa gactcaagaa ggacggggag
     1921 gtgaggggct caattaacag gttcaagaca tctgactatg tgaaagaagc aaagcagctc
     1981 ctcaaagtgc aaaaagctta ccaccagctg gaccaaagtt ttatcgacac gtacatcgat
     2041 cttttggaaa cacgacgaac gtattacgaa ggaccaggtg aaggctcacc ctttggctgg
     2101 aaagacatta aagagtggta cgaaatgttg atgggacact gtacatactt ccctgaagag
     2161 ctgcgcagcg tcaagtacgc ctataacgcc gacctgtaca acgcccttaa cgacctcaac
     2221 aatttggtga tcactagaga cgagaacgaa aagctcgagt actacgaaaa attccaaatt
     2281 atcgaaaatg tcttcaagca gaagaaaaag cctacactca aacagattgc caaagagata
     2341 ttggtaaacg aagaggatat caaagggtac agagtcactt ccaccgggaa gccggagttc
     2401 actaatctca aggtttatca cgacataaag gacataaccg cccggaaaga gatcattgag
     2461 aacgccgagc ttctggacca gattgcaaaa attcttacta tctaccaatc atccgaggac
     2521 atccaggagg agctcaccaa tcttaatagc gagctcacgc aagaagaaat tgagcaaatt
     2581 agcaatctca aggggtacac cggaacccat aatttgagcc ttaaggctat taacttgatt
     2641 ctggacgagc tttggcacac gaacgataat cagattgcca tttttaaccg gcttaaactc
     2701 gttccgaaga aagttgattt gagtcaacaa aaggaaatac ctaccacttt ggtagacgat
     2761 tttattctct cccctgtggt aaagcgatcc tttatccagt caatcaaagt gattaatgca
     2821 atcattaaaa agtacgggct gcccaatgac attattatcg agctcgcaag ggagaagaac
     2881 agcaaagatg cccagaagat gataaacgag atgcagaagc ggaatcgaca gacaaatgaa
     2941 aggattgaag aaataatccg aacaacaggg aaggaaaatg ctaagtatct tatcgaaaag
     3001 attaagctcc acgatatgca ggagggcaag tgcctttact ctctcgaggc gattccattg
     3061 gaggacctcc ttaacaatcc gttcaactac gaggtggatc acatcatccc caggagcgtg
     3121 agcttcgaca actcattcaa caacaaggtg ttagtgaagc aggaggagaa cagcaagaaa
     3181 ggtaatagga ccccattcca gtaccttagt agctctgact ccaagatcag ctacgagact
     3241 ttcaagaagc acatactgaa cttagccaag ggtaaaggcc gcattagcaa aaccaagaag
     3301 gagtatttgc tggaagaaag ggacatcaac cggttctcag tccagaagga cttcatcaac
     3361 aggaacctgg tggacacaag atacgctacc aggggcctga tgaacctctt gagatcttac
     3421 ttccgggtca ataacctgga tgttaaggtc aagtctatca acggcggctt caccagcttc
     3481 ctgagaagaa agtggaagtt caagaaggag cgaaacaagg ggtacaaaca ccatgccgag
     3541 gacgctctga taatcgctaa cgccgacttc atcttcaagg agtggaagaa gctggacaaa
     3601 gccaagaagg tgatggaaaa ccagatgttc gaagagaagc aggctgagtc catgccagag
     3661 atcgagacag agcaggaata caaggaaatc ttcatcaccc ctcaccagat caaacatatt
     3721 aaggatttca aagactataa gtactcccat agagttgaca aaaagcccaa cagagaactg
     3781 atcaacgaca ctctctactc taccagaaag gacgataaag gtaacactct tattgtcaac
     3841 aatctcaatg gcctgtacga caaggacaac gataagctga agaaactgat caacaaatca
     3901 cccgagaagc tcctgatgta ccaccacgac ccacagacat accagaagtt gaagctcatc
     3961 atggaacagt acggcgacga gaagaacccc ctgtacaaat actacgagga aacggggaat
     4021 tacctcacca agtactctaa aaaggacaac ggacccgtta ttaaaaagat caaatattat
     4081 ggtaacaagc ttaatgctca tctggacatc accgatgatt accccaattc cagaaataag
     4141 gtggtcaaac tgtctaccgg aggattcagt aaggaaagca tactgccgaa aaggaattcc
     4201 gacaagctga tcgctaggaa gaaggactgg gatcccaaaa aatacggggg attcgactcc
     4261 cccacagttg cgtacagcgt gctcgtggtg gcaaaagtgg agaaggggaa atccaaaaaa
     4321 ctaaaatcag tcaaagagct gcttggcatt acaatcatgg agaggtcttc cttcgagaag
     4381 aatccaattg acttcctcga ggctaaggga tacaaggagg tgaagaagga cttgataatc
     4441 aaactgccga agtacagctt gttcgagctc gaaaatggcc gcaagaggat gctggctagc
     4501 gctggcgagc tccagaaggg caacgagctg gccctgccca gcaagtacgt gaacttcctt
     4561 tatctcgcat cacactacga gaagctcaag ggatcacccg aggacaacga gcagaagcag
     4621 ctgtttgtgg agcagcacaa gcactatctc gatgaaatta ttgaacagat ctccgaattt
     4681 tctaaacggg taattctcgc agacgctaac ctcgacaaag tgctgagcgc atacaataaa
     4741 catcgggaca agccaattag ggagcaggct gagaacatca tccatctttt caccttgaca
     4801 aatctcggcg ctccagccgc cttcaagtac ttcgacacca ccatcgatag gaagcgatac
     4861 acatcaacga aagaggtgct ggacgccact ctgatccacc agagcatcac cggcctgtac
     4921 gaaacgagaa tcgacttgtc tcagctgggc ggagatgact acaaggacga cgacgacaag
     4981 gactataagg acgatgatga taaggattac aaagatgacg acgataagcc ccccaagaag
     5041 aagaggaagg tggaggccag ctaagaattc aataaaagat ctttattttc attagatctg
     5101 tgtgttggtt ttttgtgtgt aagaattccg gccgcaggaa cccctagtga tggagttggc
     5161 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     5221 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggatcg
     5281 gatcccgggc ccgtcgactg cagaggcctg catgcaagct tggcgtaatc atggtcatag
     5341 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     5401 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     5461 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     5521 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     5581 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     5641 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     5701 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5761 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5821 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5881 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5941 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     6001 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     6061 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     6121 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     6181 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     6241 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     6301 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     6361 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     6421 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     6481 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     6541 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     6601 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     6661 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6721 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6781 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     6841 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6901 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6961 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     7021 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     7081 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     7141 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     7201 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     7261 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     7321 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     7381 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     7441 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     7501 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtc
//
