LOCUS       sequence-417999-        7556 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Coding for SaSp Cas9 alongside Gollum sgRNA targeting GFP Target 4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7556)
  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 7556)
  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 Gollum Target 4- eCMV-...
FEATURES             Location/Qualifiers
     source          1..7556
                     /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        881..1184
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1185..1388
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1339..1359
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     RBS             3066..3074
                     /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             4958..5029
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     CDS             5033..5053
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    5072..5120
                     /note="synthetic polyadenylation signal"
     repeat_region   5137..5277
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5137..5266
                     /label=AAV2 ITR
     primer_bind     complement(5335..5351)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5335..5351)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5348..5370)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5359..5375
                     /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(5383..5389,5390..5407,5408..5413))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5428..5449
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5737..6325)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5817..5836)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6496..7287,7288..7356))
                     /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     7119..7138
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7357..7461)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7529..7547
                     /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 agctagaaat agcaagttaa aataaggcaa aatgccgtgt ttaacttgaa
      781 aaagtgtttt ttttctagag ggtaccgggg cccgcctagg tagttattaa tagtaatcaa
      841 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
      901 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
      961 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     1021 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     1081 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     1141 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
     1201 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
     1261 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
     1321 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
     1381 gcagagctgg tttagtgaac cgtcagatcc gctagcgcta ccggtcgcca ccatgaagag
     1441 gaattacatt ctgggcctgg acatcgggat cacctctgtg gggtatggca ttatcgacta
     1501 cgagacaaga gacgtgatcg acgcaggcgt cagactcttc aaggaggcca acgtggagaa
     1561 taacgaaggc aggcggagca agaggggtgc aaggagattg aaaagaaggc ggcgccacag
     1621 aatccagagg gtgaagaagc tccttttcga ctacaacttg ctcaccgatc atagcgagct
     1681 gtccggcatc aatccttacg aggctagggt caagggattg agccagaagt tgtcagagga
     1741 ggagttcagc gcagcactgc tccatcttgc caagaggaga ggagtccaca acgtgaatga
     1801 ggtggaggaa gatactggga atgagctgag cacgaaggag cagatctcta gaaactcaaa
     1861 agccttggaa gaaaagtatg ttgctgaact ccagcttgaa agactcaaga aggacgggga
     1921 ggtgaggggc tcaattaaca ggttcaagac atctgactat gtgaaagaag caaagcagct
     1981 cctcaaagtg caaaaagctt accaccagct ggaccaaagt tttatcgaca cgtacatcga
     2041 tcttttggaa acacgacgaa cgtattacga aggaccaggt gaaggctcac cctttggctg
     2101 gaaagacatt aaagagtggt acgaaatgtt gatgggacac tgtacatact tccctgaaga
     2161 gctgcgcagc gtcaagtacg cctataacgc cgacctgtac aacgccctta acgacctcaa
     2221 caatttggtg atcactagag acgagaacga aaagctcgag tactacgaaa aattccaaat
     2281 tatcgaaaat gtcttcaagc agaagaaaaa gcctacactc aaacagattg ccaaagagat
     2341 attggtaaac gaagaggata tcaaagggta cagagtcact tccaccggga agccggagtt
     2401 cactaatctc aaggtttatc acgacataaa ggacataacc gcccggaaag agatcattga
     2461 gaacgccgag cttctggacc agattgcaaa aattcttact atctaccaat catccgagga
     2521 catccaggag gagctcacca atcttaatag cgagctcacg caagaagaaa ttgagcaaat
     2581 tagcaatctc aaggggtaca ccggaaccca taatttgagc cttaaggcta ttaacttgat
     2641 tctggacgag ctttggcaca cgaacgataa tcagattgcc atttttaacc ggcttaaact
     2701 cgttccgaag aaagttgatt tgagtcaaca aaaggaaata cctaccactt tggtagacga
     2761 ttttattctc tcccctgtgg taaagcgatc ctttatccag tcaatcaaag tgattaatgc
     2821 aatcattaaa aagtacgggc tgcccaatga cattattatc gagctcgcaa gggagaagaa
     2881 cagcaaagat gcccagaaga tgataaacga gatgcagaag cggaatcgac agacaaatga
     2941 aaggattgaa gaaataatcc gaacaacagg gaaggaaaat gctaagtatc ttatcgaaaa
     3001 gattaagctc cacgatatgc aggagggcaa gtgcctttac tctctcgagg cgattccatt
     3061 ggaggacctc cttaacaatc cgttcaacta cgaggtggat cacatcatcc ccaggagcgt
     3121 gagcttcgac aactcattca acaacaaggt gttagtgaag caggaggaga acagcaagaa
     3181 aggtaatagg accccattcc agtaccttag tagctctgac tccaagatca gctacgagac
     3241 tttcaagaag cacatactga acttagccaa gggtaaaggc cgcattagca aaaccaagaa
     3301 ggagtatttg ctggaagaaa gggacatcaa ccggttctca gtccagaagg acttcatcaa
     3361 caggaacctg gtggacacaa gatacgctac caggggcctg atgaacctct tgagatctta
     3421 cttccgggtc aataacctgg atgttaaggt caagtctatc aacggcggct tcaccagctt
     3481 cctgagaaga aagtggaagt tcaagaagga gcgaaacaag gggtacaaac accatgccga
     3541 ggacgctctg ataatcgcta acgccgactt catcttcaag gagtggaaga agctggacaa
     3601 agccaagaag gtgatggaaa accagatgtt cgaagagaag caggctgagt ccatgccaga
     3661 gatcgagaca gagcaggaat acaaggaaat cttcatcacc cctcaccaga tcaaacatat
     3721 taaggatttc aaagactata agtactccca tagagttgac aaaaagccca acagagaact
     3781 gatcaacgac actctctact ctaccagaaa ggacgataaa ggtaacactc ttattgtcaa
     3841 caatctcaat ggcctgtacg acaaggacaa cgataagctg aagaaactga tcaacaaatc
     3901 acccgagaag ctcctgatgt accaccacga cccacagaca taccagaagt tgaagctcat
     3961 catggaacag tacggcgacg agaagaaccc cctgtacaaa tactacgagg aaacggggaa
     4021 ttacctcacc aagtactcta aaaaggacaa cggacccgtt attaaaaaga tcaaatatta
     4081 tggtaacaag cttaatgctc atctggacat caccgatgat taccccaatt ccagaaataa
     4141 ggtggtcaaa ctgtctaccg gaggattcag taaggaaagc atactgccga aaaggaattc
     4201 cgacaagctg atcgctagga agaaggactg ggatcccaaa aaatacgggg gattcgactc
     4261 ccccacagtt gcgtacagcg tgctcgtggt ggcaaaagtg gagaagggga aatccaaaaa
     4321 actaaaatca gtcaaagagc tgcttggcat tacaatcatg gagaggtctt ccttcgagaa
     4381 gaatccaatt gacttcctcg aggctaaggg atacaaggag gtgaagaagg acttgataat
     4441 caaactgccg aagtacagct tgttcgagct cgaaaatggc cgcaagagga tgctggctag
     4501 cgctggcgag ctccagaagg gcaacgagct ggccctgccc agcaagtacg tgaacttcct
     4561 ttatctcgca tcacactacg agaagctcaa gggatcaccc gaggacaacg agcagaagca
     4621 gctgtttgtg gagcagcaca agcactatct cgatgaaatt attgaacaga tctccgaatt
     4681 ttctaaacgg gtaattctcg cagacgctaa cctcgacaaa gtgctgagcg catacaataa
     4741 acatcgggac aagccaatta gggagcaggc tgagaacatc atccatcttt tcaccttgac
     4801 aaatctcggc gctccagccg ccttcaagta cttcgacacc accatcgata ggaagcgata
     4861 cacatcaacg aaagaggtgc tggacgccac tctgatccac cagagcatca ccggcctgta
     4921 cgaaacgaga atcgacttgt ctcagctggg cggagatgac tacaaggacg acgacgacaa
     4981 ggactataag gacgatgatg ataaggatta caaagatgac gacgataagc cccccaagaa
     5041 gaagaggaag gtggaggcca gctaagaatt caataaaaga tctttatttt cattagatct
     5101 gtgtgttggt tttttgtgtg taagaattcc ggccgcagga acccctagtg atggagttgg
     5161 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
     5221 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggatc
     5281 ggatcccggg cccgtcgact gcagaggcct gcatgcaagc ttggcgtaat catggtcata
     5341 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     5401 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     5461 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5521 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     5581 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5641 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     5701 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5761 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5821 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     5881 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     5941 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     6001 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     6061 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     6121 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     6181 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     6241 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     6301 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     6361 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     6421 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     6481 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     6541 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6601 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     6661 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     6721 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6781 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     6841 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     6901 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     6961 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     7021 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     7081 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     7141 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     7201 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     7261 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     7321 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     7381 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     7441 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     7501 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc
//
