LOCUS       sequence-391672-        6832 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid with CCR5 target sites for ITPN or HMEJ knock-in at 
            the human CCR5 safe harbour locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6832)
  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 6832)
  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: BB44_pmc.DonorR5.TS
FEATURES             Location/Qualifiers
     source          1..6832
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     132..149
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    266..287
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(302..307,308..325,326..332)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    340..356
                     /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)."
     primer_bind     345..367
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     364..380
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     364..380
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1759..2268
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2188..2206
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      2275..2284
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2281..2991
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2426..2444)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2694..2713
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3018..3035)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3024..3248
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    3628..3639
                     /label=ZF binding site
                     /bound_moiety="CCR5TC zinc-finger domain"
                     /note="target sequence for the CCR5TC zinc-finger domain 
                     from the CCR5-224 (+) zinc-finger nuclease (Perez et al., 
                     2008; Gross et al., 2013)"
     primer_bind     complement(4596..4613)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4596..4612)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4605..4627)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4821..4840)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4940..4962
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5000..5018)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5086..5190
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5191..5259,5260..6051)
                     /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     complement(5409..5428)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6222..6810
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6711..6730
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
       61 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
      121 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
      181 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
      241 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
      301 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
      361 acacaggaaa cagctatgac catgattacg ccaagcttgc atgcaggcct ctgcagtcga
      421 cgggcccggg atccgatacg acgcggccgc agatggtgct ttcatgaatt cccccaacag
      481 agccaagctc tccatctagt ggacagggaa gctagcagca aaatttcgaa tgcatctagt
      541 gtttcatggc acagttagta tataattctt tatggttcaa aattaaaaat gagcttttct
      601 aggggcttct ctcagctgcc tagtctaagg tgcagggagt ttgagactca cagggtttaa
      661 taagagaaaa ttctcagcta gagcagctga acttaaatag actaggcaag acagctggtt
      721 ataagactaa actacccaga atgcatgaca ttcatctgtg gtggcagacg aaacattttt
      781 tattatatta tttcttgggt atgtatgaca actcttaatt gtggcaactc agaaactaca
      841 aacacaaact tcacagaaaa tgtgaggatt ttacaattgg ctgttgtcat ctatgacctt
      901 ccctgggact tgggcacccg gccatttcac tctgactaca tcatgtcacc aaacatctga
      961 tggtcttgcc ttttaattct cttttcgagg actgagaggg agggtagcat ggtagttaag
     1021 agtgcaggct tcccgcattc aaaatcggtt gcttactagc tgtgtggctt tgagcaagtt
     1081 actcaccctc tctgtgcttc aaggtccttg tctgcaaaat gtgaaaaata tttcctgcct
     1141 cataaggttg ccctaaggat taaatgaatg aatgggtatg atgcttagaa cagtgattgg
     1201 catccagtat gtgccctcga ggcctcttaa ttattactgg cttgctcata gtgcatgttc
     1261 tttgtgggct aactctagcg tcaataaaaa tgttaagact gagttgcagc cgggcatggt
     1321 ggctcatgcc tgtaatccca gcattctagg aggctgaggc aggaggatcg cttgagccca
     1381 ggagttcgag accagcctgg gcaacatagt gtgatcttgt atctataaaa ataaacaaaa
     1441 ttagcttggt gtggtggcgc ctgtagtccc cagccacttg gaggggtgag gtgagaggat
     1501 tgcttgagcc cgggatggtc caggctgcag tgagccatga tcgtgccact gcactccagc
     1561 ctgggcgaca gagtgagacc ctgtctcaca acaacaacaa caacaacaaa aaggctgagc
     1621 tgcaccatgc ttgacccagt ttcttaaaat tgttgtcaaa gcttcattca ctccatggtg
     1681 ctatagagca caagatttta tttggtgaga tggtgctttc atgaattccc ccaacagagc
     1741 aagcttgtta gcttccacgg ggttggggtt gcgccttttc caaggcagcc ctgggtttgc
     1801 gcagggacgc ggctgctctg ggcgtggttc cgggaaacgc agcggcgccg accctgggtc
     1861 tcgcacattc ttcacgtccg ttcgcagcgt cacccggatc ttcgccgcta cccttgtggg
     1921 ccccccggcg acgcttcctg ctccgcccct aagtcgggaa ggttccttgc ggttcgcggc
     1981 gtgccggacg tgacaaacgg aagccgcacg tctcactagt accctcgcag acggacagcg
     2041 ccagggagca atggcagcgc gccgaccgcg atgggctgtg gccaatagcg gctgctcagc
     2101 agggcgcgcc gagagcagcg gccgggaagg ggcggtgcgg gaggcggggt gtggggcggt
     2161 agtgtgggcc ctgttcctgc ccgcgcggtg ttccgcattc tgcaagcctc cggagcgcac
     2221 gtcggcagtc ggctccctcg ttgaccgaat caccgacctc tctccccaag atctgccacc
     2281 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag
     2341 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
     2401 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
     2461 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac
     2521 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc
     2581 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac
     2641 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
     2701 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc
     2761 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct
     2821 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc
     2881 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa
     2941 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta aagcggccgc
     3001 gtcgagtcta ggatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     3061 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     3121 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     3181 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     3241 ctctatggaa gctaacacgc gtctccatct agtggacagg gaagctagca gcaaaccttc
     3301 ccttcactac aaaacttcat tgcttggcca aaaagagagt taattcaatg tagacatcta
     3361 tgtaggcaat taaaaaccta ttgatgtata aaacagtttg cattcatgga gggcaactaa
     3421 atacattcta ggactttata aaagatcact ttttatttat gcacagggtg gaacaagatg
     3481 gattatcaag tgtcaagtcc aatctatgac atcaattatt atacatcgga gccctgccaa
     3541 aaaatcaatg tgaagcaaat cgcagcccgc ctcctgcctc cgctctactc actggtgttc
     3601 atctttggtt ttgtgggcaa catgctggtc atcctcatcc tgataaactg caaaaggctg
     3661 aagagcatga ctgacatcta cctgctcaac ctggccatct ctgacctgtt tttccttctt
     3721 actgtcccct tctgggctca ctatgctgcc gcccagtggg actttggaaa tacaatgtgt
     3781 caactcttga cagggctcta ttttataggc ttcttctctg gaatcttctt catcatcctc
     3841 ctgacaatcg ataggtacct ggctgtcgtc catgctgtgt ttgctttaaa agccaggacg
     3901 gtcacctttg gggtggtgac aagtgtgatc acttgggtgg tggctgtgtt tgcgtctctc
     3961 ccaggaatca tctttaccag atctcaaaaa gaaggtcttc attacacctg cagctctcat
     4021 tttccataca gtcagtatca attctggaag aatttccaga cattaaagat agtcatcttg
     4081 gggctggtcc tgccgctgct tgtcatggtc atctgctact cgggaatcct aaaaactctg
     4141 cttcggtgtc gaaatgagaa gaagaggcac agggctgtga ggcttatctt caccatcatg
     4201 attgtttatt ttctcttctg ggctccctac aacattgtcc ttctcctgaa caccttccag
     4261 gaattctttg gcctgaataa ttgcagtagc tctaacaggt tggaccaagc tatgcaggtg
     4321 acagagactc ttgggatgac gcactgctgc atcaacccca tcatctatgc ctttgtcggg
     4381 gagaagttca gaaactacct cttagtcttc ttccaaaagc acattgccaa acgcttctgc
     4441 aaatgctgtt ctattttcca gccctagata aacagatggt gctttcatga attcccccaa
     4501 cagagccaag ctctccatct agtggacagg gaagctagca gcaaacgccg gcggtcgtca
     4561 tctagatgca ttcgcgaggt accgagctcg aattcactgg ccgtcgtttt acaacgtcgt
     4621 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     4681 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     4741 aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     4801 cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     4861 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     4921 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     4981 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     5041 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     5101 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     5161 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     5221 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     5281 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     5341 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     5401 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     5461 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     5521 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     5581 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     5641 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     5701 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     5761 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     5821 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     5881 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     5941 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     6001 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     6061 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     6121 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     6181 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     6241 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     6301 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     6361 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     6421 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     6481 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     6541 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     6601 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     6661 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     6721 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     6781 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tt
//
