LOCUS       sequence-391392-        5899 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid with CLYBL target sites for ITPN or HMEJ knock-in at 
            the human CLYBL safe harbor locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5899)
  AUTHORS   Wang Q, Liu J, Janssen JM, Goncalves MAFV
  TITLE     Precise homology-directed installation of large genomic edits in 
            human cells with cleaving and nicking high-specificity Cas9 
            variants.
  JOURNAL   Nucleic Acids Res. 2023 Mar 17:gkad165. doi: 10.1093/nar/gkad165.
  PUBMED    36928106
REFERENCE   2  (bases 1 to 5899)
  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: "Nucleic 
            Acids Res. 2023 Mar 17:gkad165. doi: 10.1093/nar/gkad165."
COMMENT     Sequence Label: AD59_pEP.DonorCLYBL.TS
FEATURES             Location/Qualifiers
     source          1..5899
                     /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"
     promoter        968..1477
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1397..1415
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1500..2096
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1500..1519)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1996..2016
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2106..2159
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     regulatory      2166..2175
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2172..2174,2175..2177,2178..2891)
                     /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(2172..2174,2175..2177,2178..2888)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2217..2238)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2478..2497)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2825..2846
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2918..2935)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2924..3148
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3678..3694)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3678..3694)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3691..3713)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3702..3718
                     /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(3726..3732,3733..3750,3751..3756))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3771..3792
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3909..3926)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4080..4668)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4160..4179)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4839..5630,5631..5699))
                     /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     5462..5481
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5700..5804)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5872..5890
                     /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 tatcggatcc cgggcccgtc gactgcagag ggcggccgcc agctgtgtgg
      481 ggctcgtgct cctacatacc gttatcctgt ggtagagttt taccgtggac cagatttaaa
      541 tattcagaat gctttaatgg agtaagaagg gccactgctt agagtggggc tgaagagaaa
      601 agaaagaagg agaccctgcg gaagcggcaa taccaggaag gcagttgtat gcacatcatg
      661 cagaccagga gtagcctagg ggtaggggac atgcctgggc tggacagtgg aatttgtggg
      721 aaatcagatt tccctgagca aaaaccagtg ggcagcagcc aaactggtga gcaagaactg
      781 gctgtggtgg ctgggcccga attgggagct acacctgccc gggcaggtgt agctgcgagg
      841 gaggtctctg ccaacaactc agctgtgccg tgcctgctga acggcctcac cttggcctct
      901 ggctctgttg cagctgtgtg gggctcgtgc tcctacatac cacgcgtgtt cgcgtgttag
      961 cttccacggg gttggggttg cgccttttcc aaggcagccc tgggtttgcg cagggacgcg
     1021 gctgctctgg gcgtggttcc gggaaacgca gcggcgccga ccctgggtct cgcacattct
     1081 tcacgtccgt tcgcagcgtc acccggatct tcgccgctac ccttgtgggc cccccggcga
     1141 cgcttcctgc tccgccccta agtcgggaag gttccttgcg gttcgcggcg tgccggacgt
     1201 gacaaacgga agccgcacgt ctcactagta ccctcgcaga cggacagcgc cagggagcaa
     1261 tggcagcgcg ccgaccgcga tgggctgtgg ccaatagcgg ctgctcagca gggcgcgccg
     1321 agagcagcgg ccgggaaggg gcggtgcggg aggcggggtg tggggcggta gtgtgggccc
     1381 tgttcctgcc cgcgcggtgt tccgcattct gcaagcctcc ggagcgcacg tcggcagtcg
     1441 gctccctcgt tgaccgaatc accgacctct ctccccaccg ggtacgtcgc tagcccacca
     1501 tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc cgggccgtac
     1561 gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gacccggacc
     1621 gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca
     1681 tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga
     1741 gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt
     1801 cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc
     1861 ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag ggtctgggca
     1921 gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg
     1981 agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg
     2041 acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgccggat
     2101 cgggagaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat cctggcccac
     2161 cggtcgccac catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     2221 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
     2281 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     2341 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
     2401 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     2461 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     2521 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     2581 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     2641 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     2701 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     2761 ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg
     2821 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     2881 tgtacaagta aagcggccgc gtcgagtcta ggatcagcct cgactgtgcc ttctagttgc
     2941 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     3001 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     3061 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     3121 catgctgggg atgcggtggg ctctatggaa gctaacaagc taacaagctt gttatcctgt
     3181 ggtagagttt taccgtggac cagagccatc tagaggacat tgagttaccc acatactttc
     3241 tgtgggagag gccaaggagt caggggggga ggccgggacc tgaagatcaa ctctttactg
     3301 cagagatccc tccctccctc tctctgtccc atggatccag agggaagttg aggacagttt
     3361 catgccacat gcttgacccg actcaactga gtaactgtgg ccactgagaa gactctgtcc
     3421 ttgcaaataa ctctccttag tgcccatatt ctgcagaatg tttgagtggc acgagtactt
     3481 cattcgtcta gaaatatttc atggtagtca gttgctaata acaggatatc tgcaggtagt
     3541 gtgttctcag gaggcacacc tgaagcctca gtttaaacca gctgtgtggg gctcgtgctc
     3601 ctacataccg ttatcctgtg gtagagtttt accgtggacc agcgccggcg cctgcatgca
     3661 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     3721 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     3781 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     3841 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     3901 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     3961 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     4021 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4081 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4141 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4201 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4261 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4321 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4381 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4441 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     4501 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     4561 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     4621 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     4681 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     4741 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     4801 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     4861 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     4921 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     4981 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     5041 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     5101 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     5161 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     5221 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     5281 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     5341 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     5401 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     5461 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     5521 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     5581 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     5641 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     5701 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     5761 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     5821 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     5881 atcacgaggc cctttcgtc
//
