LOCUS       sequence-391621-        5217 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 5217)
  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 5217)
  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: AZ64_pE.DonorCLYBL.TS
FEATURES             Location/Qualifiers
     source          1..5217
                     /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"
     regulatory      1484..1493
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1490..1492,1493..1495,1496..2209)
                     /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(1490..1492,1493..1495,1496..2206)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1535..1556)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1796..1815)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2143..2164
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2236..2253)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2242..2466
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2996..3012)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2996..3012)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3009..3031)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3020..3036
                     /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(3044..3050,3051..3068,3069..3074))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3089..3110
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3227..3244)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3398..3986)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3478..3497)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4157..4948,4949..5017))
                     /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     4780..4799
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5018..5122)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5190..5208
                     /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 gtcgccacca tggtgagcaa
     1501 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     1561 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     1621 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     1681 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
     1741 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     1801 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     1861 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     1921 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
     1981 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     2041 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     2101 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     2161 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaaa gcggccgcgt
     2221 cgagtctagg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     2281 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     2341 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     2401 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     2461 ctatggaagc taacaagcta acaagcttgt tatcctgtgg tagagtttta ccgtggacca
     2521 gagccatcta gaggacattg agttacccac atactttctg tgggagaggc caaggagtca
     2581 gggggggagg ccgggacctg aagatcaact ctttactgca gagatccctc cctccctctc
     2641 tctgtcccat ggatccagag ggaagttgag gacagtttca tgccacatgc ttgacccgac
     2701 tcaactgagt aactgtggcc actgagaaga ctctgtcctt gcaaataact ctccttagtg
     2761 cccatattct gcagaatgtt tgagtggcac gagtacttca ttcgtctaga aatatttcat
     2821 ggtagtcagt tgctaataac aggatatctg caggtagtgt gttctcagga ggcacacctg
     2881 aagcctcagt ttaaaccagc tgtgtggggc tcgtgctcct acataccgtt atcctgtggt
     2941 agagttttac cgtggaccag cgccggcgcc tgcatgcaag cttggcgtaa tcatggtcat
     3001 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     3061 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     3121 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     3181 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     3241 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     3301 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     3361 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     3421 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     3481 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     3541 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     3601 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     3661 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     3721 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     3781 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     3841 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     3901 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     3961 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     4021 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     4081 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     4141 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     4201 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     4261 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     4321 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     4381 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     4441 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     4501 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     4561 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     4621 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     4681 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     4741 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     4801 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     4861 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     4921 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     4981 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     5041 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     5101 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     5161 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtc
//
