LOCUS       sequence-476829-        8244 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cytosine base editor with codon-optimized S. aureus KKH Cas9 and 
            rAPOBEC1 deaminase. EGFP can be used as a transfection marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8244)
  TITLE     Elucidating the genetic mechanisms governing cytosine base editing 
            outcomes through CRISPRi screens
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8244)
  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            "Unpublished"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: pSG1039-rA1-SaKKH_P2A_EGFP
FEATURES             Location/Qualifiers
     source          1..8244
                     /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             2120..2803
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from 
                     rat"
                     /label=APOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGG
                     RHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLS
                     RYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNE
                     AHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHIL
                     WATGLK"
     CDS             6101..6121
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6131..6187
                     /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(6188..6190,6191..6193,6194..6907)
                     /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(6188..6190,6191..6193,6194..6904)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6233..6254)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6494..6513)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6841..6862
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             6913..6930
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6953..6970)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6959..7183
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(7254..7270)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7254..7270)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7267..7289)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7278..7294
                     /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(7302..7308,7309..7326,7327..7332))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7347..7368
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7485..7502)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7656..8244)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7736..7755)
                     /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 cggaaagtca gctcagagac tggcccagtg gctgtggacc ccacattgag
     2161 acggcggatc gagccccatg agtttgaggt attcttcgat ccgagagagc tccgcaagga
     2221 gacctgcctg ctttacgaaa ttaattgggg gggccggcac tccatttggc gacatacatc
     2281 acagaacact aacaagcacg tcgaagtcaa cttcatcgag aagttcacga cagaaagata
     2341 tttctgtccg aacacaaggt gcagcattac ctggtttctc agctggagcc catgcggcga
     2401 atgtagtagg gccatcactg aattcctgtc aaggtatccc cacgtcactc tgtttattta
     2461 catcgcaagg ctgtaccacc acgctgaccc ccgcaatcga caaggcctgc gggatttgat
     2521 ctcttcaggt gtgactatcc aaattatgac tgagcaggag tcaggatact gctggagaaa
     2581 ctttgtgaat tatagcccga gtaatgaagc ccactggcct aggtatcccc atctgtgggt
     2641 acgactgtac gttcttgaac tgtactgcat catactgggc ctgcctcctt gtctcaacat
     2701 tctgagaagg aagcagccac agctgacatt ctttaccatc gctcttcagt cttgtcatta
     2761 ccagcgactg cccccacaca ttctctgggc caccgggttg aaatccggag gatctagcgg
     2821 aggctcctct ggctctgaga cacctggcac aagcgagagc gcaacacctg aaagcagcgg
     2881 gggcagcagc ggggggtcag ggaagcgaaa ttacattctg gggctggcca ttggcattac
     2941 atcagtgggc tatggcatca ttgactacga gacaagggac gtgatcgacg ccggcgtgag
     3001 actgttcaag gaggccaacg tggagaacaa tgagggccgg agatccaaga ggggagcaag
     3061 gcgcctgaag cggagaaggc gccacagaat ccagagagtg aagaagctgc tgttcgatta
     3121 caacctgctg accgaccact ccgagctgtc tggcatcaat ccttatgagg ccagagtgaa
     3181 gggcctgtcc cagaagctgt ctgaggagga gtttagcgcc gccctgctgc acctggcaaa
     3241 gaggagaggc gtgcacaacg tgaatgaggt ggaggaggac accggcaacg agctgtccac
     3301 aaaggagcag atcagccgca attccaaggc cctggaggag aagtatgtgg ccgagctgca
     3361 gctggagcgg ctgaagaagg atggcgaggt gaggggctcc atcaatcgct tcaagacctc
     3421 tgactacgtg aaggaggcca agcagctgct gaaggtgcag aaggcctacc accagctgga
     3481 tcagtccttt atcgatacat atatcgacct gctggagaca aggcgcacat actatgaggg
     3541 accaggagag ggctctccct tcggctggaa ggacatcaag gagtggtacg agatgctgat
     3601 gggccactgc acctattttc cagaggagct gagaagcgtg aagtacgcct ataacgccga
     3661 tctgtacaac gccctgaatg acctgaacaa cctggtcatc accagggatg agaacgagaa
     3721 gctggagtac tatgagaagt tccagatcat cgagaacgtg ttcaagcaga agaagaagcc
     3781 tacactgaag cagatcgcca aggagatcct ggtgaacgag gaggacatca agggctaccg
     3841 cgtgacctcc acaggcaagc cagagttcac caatctgaag gtgtatcacg atatcaagga
     3901 catcacagcc cggaaggaga tcatcgagaa cgccgagctg ctggatcaga tcgccaagat
     3961 cctgaccatc tatcagagct ccgaggacat ccaggaggag ctgaccaacc tgaatagcga
     4021 gctgacacag gaggagatcg agcagatcag caatctgaag ggctacaccg gcacacacaa
     4081 cctgagcctg aaggccatca atctgatcct ggatgagctg tggcacacaa acgacaatca
     4141 gatcgccatc tttaaccggc tgaagctggt gccaaagaag gtggacctgt cccagcagaa
     4201 ggagatccca accacactgg tggacgattt catcctgtct cccgtggtga agcggagctt
     4261 catccagagc atcaaagtga tcaacgccat catcaagaag tacggcctgc ccaatgatat
     4321 catcatcgag ctggccaggg agaagaactc caaggacgcc cagaagatga tcaatgagat
     4381 gcagaagagg aaccgccaga ccaatgagcg gatcgaggag atcatcagaa ccacaggcaa
     4441 ggagaacgcc aagtacctga tcgagaagat caagctgcac gatatgcagg agggcaagtg
     4501 tctgtattct ctggaggcca tccctctgga ggacctgctg aacaatccat tcaactacga
     4561 ggtggatcac atcatccccc ggagcgtgag cttcgacaat tcttttaaca ataaggtgct
     4621 ggtgaagcag gaggagaaca gcaagaaggg caataggacc cctttccagt acctgtctag
     4681 ctccgattct aagatcagct acgagacatt caagaagcac atcctgaatc tggccaaggg
     4741 caagggccgc atcagcaaga ccaagaagga gtacctgctg gaggagcggg acatcaacag
     4801 attctccgtg cagaaggact tcatcaaccg gaatctggtg gacaccagat acgccacacg
     4861 cggcctgatg aatctgctgc ggtcttattt cagagtgaac aatctggatg tgaaggtgaa
     4921 gagcatcaac ggcggcttca cctcctttct gcggagaaag tggaagttta agaaggagcg
     4981 caacaagggc tataagcacc acgccgagga tgccctgatc atcgccaatg ccgacttcat
     5041 ctttaaggag tggaagaagc tggacaaggc caagaaagtg atggagaacc agatgttcga
     5101 ggagaagcag gccgagagca tgcccgagat cgagacagag caggagtaca aggagatttt
     5161 catcacacct caccagatca agcacatcaa ggacttcaag gactacaagt attctcacag
     5221 ggtggataag aagcccaacc gcaagctgat caatgacacc ctgtatagca cacggaagga
     5281 cgataagggc aataccctga tcgtgaacaa tctgaacggc ctgtacgaca aggataatga
     5341 caagctgaag aagctgatca acaagtctcc cgagaagctg ctgatgtacc accacgatcc
     5401 tcagacatat cagaagctga agctgatcat ggagcagtac ggcgacgaga agaacccact
     5461 gtataagtac tatgaggaga caggcaacta cctgacaaag tatagcaaga aggataatgg
     5521 ccccgtgatc aagaagatca agtactatgg caacaagctg aatgcccacc tggacatcac
     5581 cgacgattac cctaactctc gcaataaggt ggtgaagctg agcctgaagc cataccggtt
     5641 cgacgtgtac ctggacaacg gcgtgtataa gtttgtgaca gtgaagaatc tggatgtgat
     5701 caagaaggag aactactatg aggtgaacag caagtgctac gaggaggcca agaagctgaa
     5761 gaagatcagc aaccaggccg agttcatcgc ctctttttac aagaatgacc tgatcaagat
     5821 caatggcgag ctgtatagag tgatcggcgt gaacaatgat ctgctgaaca gaatcgaagt
     5881 gaatatgatc gacatcacct acagggagta tctggagaac atgaatgata agaggccccc
     5941 tcatatcatc aagaccatcg cctctaagac acagagcatc aagaagtaca gcacagacat
     6001 cctggggaac ctgtatgaag tcaagagcaa gaaacatcct cagattatca agaaaggctc
     6061 tggcggctca aaaagaaccg ccgacggcag cgaattcgag cccaagaaga agaggaaagt
     6121 cggaagcgga gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc
     6181 tggacctatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     6241 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
     6301 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     6361 gcccaccctc gtgaccaccc tgacctatgg agtgcagtgc ttcagccgct accccgacca
     6421 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     6481 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     6541 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     6601 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     6661 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     6721 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     6781 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca
     6841 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     6901 caagtaaccg gtcatcatca ccatcaccat tgagtttaaa cccgctgatc agcctcgact
     6961 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     7021 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     7081 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     7141 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     7201 accagctggg gctcgatacc gtcgacctct agctagagct tggcgtaatc atggtcatag
     7261 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     7321 ataaagtgta aagcctaggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     7381 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     7441 cgcgcgggga gaggcggttt gcgtattggg cgcacttccg cttcctcgct cactgactcg
     7501 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     7561 ttatccacag aatcagagga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     7621 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     7681 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     7741 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     7801 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     7861 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     7921 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     7981 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     8041 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     8101 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     8161 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     8221 acgcgcagaa aaaaaggatc tcaa
//
