LOCUS       sequence-476832-        7581 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Codon-optimized S. aureus KKH Cas9. 2x BsaI sites for easy cloning 
            of varying deaminases. EGFP can serve as a transfection marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7581)
  TITLE     Elucidating the genetic mechanisms governing cytosine base editing 
            outcomes through CRISPRi screens
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7581)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pSG1016-2xBsaI-SaKKH_P2A_EGFP
FEATURES             Location/Qualifiers
     source          1..7581
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /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     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1215..1234)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1406..1785
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1786..1989
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1940..1960
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2031..2050
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2031..2049
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2099..2119
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5438..5458
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5468..5524
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(5525..5527,5528..5530,5531..6244)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(5525..5527,5528..5530,5531..6241)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5570..5591)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5831..5850)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6178..6199
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             6250..6267
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6290..6307)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6296..6520
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6591..6607)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6591..6607)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6604..6626)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6615..6631
                     /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(6639..6645,6646..6663,6664..6669))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6684..6705
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6822..6839)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6993..7581)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7073..7092)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacactcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gggacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atcgatctcc cgatccccta
     1201 gggtcgactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
     1261 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa
     1321 ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc
     1381 gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca
     1441 attacggggt cattagttca tagcccatat attgagttcc gcgttacata acttacggta
     1501 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     1561 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     1621 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     1681 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     1741 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
     1801 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
     1861 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
     1921 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
     1981 agcagagctg gtttagtgaa ccgtcagatc cgctagagat ccgcggccgc taatacgact
     2041 cactataggg agagccgcca ccatgaaacg gacagccgac ggaagcgagt tcgagtcacc
     2101 aaagaagaag cggaaagtct gagaccatta atgggtctct tccggaggat ctagcggagg
     2161 ctcctctggc tctgagacac ctggcacaag cgagagcgca acacctgaaa gcagcggggg
     2221 cagcagcggg gggtcaggga agcgaaatta cattctgggg ctggccattg gcattacatc
     2281 agtgggctat ggcatcattg actacgagac aagggacgtg atcgacgccg gcgtgagact
     2341 gttcaaggag gccaacgtgg agaacaatga gggccggaga tccaagaggg gagcaaggcg
     2401 cctgaagcgg agaaggcgcc acagaatcca gagagtgaag aagctgctgt tcgattacaa
     2461 cctgctgacc gaccactccg agctgtctgg catcaatcct tatgaggcca gagtgaaggg
     2521 cctgtcccag aagctgtctg aggaggagtt tagcgccgcc ctgctgcacc tggcaaagag
     2581 gagaggcgtg cacaacgtga atgaggtgga ggaggacacc ggcaacgagc tgtccacaaa
     2641 ggagcagatc agccgcaatt ccaaggccct ggaggagaag tatgtggccg agctgcagct
     2701 ggagcggctg aagaaggatg gcgaggtgag gggctccatc aatcgcttca agacctctga
     2761 ctacgtgaag gaggccaagc agctgctgaa ggtgcagaag gcctaccacc agctggatca
     2821 gtcctttatc gatacatata tcgacctgct ggagacaagg cgcacatact atgagggacc
     2881 aggagagggc tctcccttcg gctggaagga catcaaggag tggtacgaga tgctgatggg
     2941 ccactgcacc tattttccag aggagctgag aagcgtgaag tacgcctata acgccgatct
     3001 gtacaacgcc ctgaatgacc tgaacaacct ggtcatcacc agggatgaga acgagaagct
     3061 ggagtactat gagaagttcc agatcatcga gaacgtgttc aagcagaaga agaagcctac
     3121 actgaagcag atcgccaagg agatcctggt gaacgaggag gacatcaagg gctaccgcgt
     3181 gacctccaca ggcaagccag agttcaccaa tctgaaggtg tatcacgata tcaaggacat
     3241 cacagcccgg aaggagatca tcgagaacgc cgagctgctg gatcagatcg ccaagatcct
     3301 gaccatctat cagagctccg aggacatcca ggaggagctg accaacctga atagcgagct
     3361 gacacaggag gagatcgagc agatcagcaa tctgaagggc tacaccggca cacacaacct
     3421 gagcctgaag gccatcaatc tgatcctgga tgagctgtgg cacacaaacg acaatcagat
     3481 cgccatcttt aaccggctga agctggtgcc aaagaaggtg gacctgtccc agcagaagga
     3541 gatcccaacc acactggtgg acgatttcat cctgtctccc gtggtgaagc ggagcttcat
     3601 ccagagcatc aaagtgatca acgccatcat caagaagtac ggcctgccca atgatatcat
     3661 catcgagctg gccagggaga agaactccaa ggacgcccag aagatgatca atgagatgca
     3721 gaagaggaac cgccagacca atgagcggat cgaggagatc atcagaacca caggcaagga
     3781 gaacgccaag tacctgatcg agaagatcaa gctgcacgat atgcaggagg gcaagtgtct
     3841 gtattctctg gaggccatcc ctctggagga cctgctgaac aatccattca actacgaggt
     3901 ggatcacatc atcccccgga gcgtgagctt cgacaattct tttaacaata aggtgctggt
     3961 gaagcaggag gagaacagca agaagggcaa taggacccct ttccagtacc tgtctagctc
     4021 cgattctaag atcagctacg agacattcaa gaagcacatc ctgaatctgg ccaagggcaa
     4081 gggccgcatc agcaagacca agaaggagta cctgctggag gagcgggaca tcaacagatt
     4141 ctccgtgcag aaggacttca tcaaccggaa tctggtggac accagatacg ccacacgcgg
     4201 cctgatgaat ctgctgcggt cttatttcag agtgaacaat ctggatgtga aggtgaagag
     4261 catcaacggc ggcttcacct cctttctgcg gagaaagtgg aagtttaaga aggagcgcaa
     4321 caagggctat aagcaccacg ccgaggatgc cctgatcatc gccaatgccg acttcatctt
     4381 taaggagtgg aagaagctgg acaaggccaa gaaagtgatg gagaaccaga tgttcgagga
     4441 gaagcaggcc gagagcatgc ccgagatcga gacagagcag gagtacaagg agattttcat
     4501 cacacctcac cagatcaagc acatcaagga cttcaaggac tacaagtatt ctcacagggt
     4561 ggataagaag cccaaccgca agctgatcaa tgacaccctg tatagcacac ggaaggacga
     4621 taagggcaat accctgatcg tgaacaatct gaacggcctg tacgacaagg ataatgacaa
     4681 gctgaagaag ctgatcaaca agtctcccga gaagctgctg atgtaccacc acgatcctca
     4741 gacatatcag aagctgaagc tgatcatgga gcagtacggc gacgagaaga acccactgta
     4801 taagtactat gaggagacag gcaactacct gacaaagtat agcaagaagg ataatggccc
     4861 cgtgatcaag aagatcaagt actatggcaa caagctgaat gcccacctgg acatcaccga
     4921 cgattaccct aactctcgca ataaggtggt gaagctgagc ctgaagccat accggttcga
     4981 cgtgtacctg gacaacggcg tgtataagtt tgtgacagtg aagaatctgg atgtgatcaa
     5041 gaaggagaac tactatgagg tgaacagcaa gtgctacgag gaggccaaga agctgaagaa
     5101 gatcagcaac caggccgagt tcatcgcctc tttttacaag aatgacctga tcaagatcaa
     5161 tggcgagctg tatagagtga tcggcgtgaa caatgatctg ctgaacagaa tcgaagtgaa
     5221 tatgatcgac atcacctaca gggagtatct ggagaacatg aatgataaga ggccccctca
     5281 tatcatcaag accatcgcct ctaagacaca gagcatcaag aagtacagca cagacatcct
     5341 ggggaacctg tatgaagtca agagcaagaa acatcctcag attatcaaga aaggctctgg
     5401 cggctcaaaa agaaccgccg acggcagcga attcgagccc aagaagaaga ggaaagtcgg
     5461 aagcggagct actaacttca gcctgctgaa gcaggctgga gacgtggagg agaaccctgg
     5521 acctatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     5581 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
     5641 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     5701 caccctcgtg accaccctga cctatggagt gcagtgcttc agccgctacc ccgaccacat
     5761 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     5821 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     5881 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     5941 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     6001 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     6061 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     6121 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     6181 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     6241 gtaaccggtc atcatcacca tcaccattga gtttaaaccc gctgatcagc ctcgactgtg
     6301 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     6361 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     6421 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     6481 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     6541 agctggggct cgataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     6601 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     6661 aagtgtaaag cctagggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     6721 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     6781 gcggggagag gcggtttgcg tattgggcgc acttccgctt cctcgctcac tgactcgctg
     6841 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     6901 tccacagaat cagaggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     6961 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     7021 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     7081 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     7141 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     7201 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     7261 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     7321 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     7381 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     7441 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     7501 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     7561 cgcagaaaaa aaggatctca a
//
