LOCUS       sequence-462769-        7260 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV production plasmid for UbC-tEPOR-2A-YFP vector from Fig. 2 that 
            mediates HDR at CCR5 locus using CCR5-sg3 gRNA. YFP is followed by a
            BGH polyA. AAV genome is flanked by AAV2 ITRs..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7260)
  AUTHORS   Luna SE, Camarena J, Hampton JP, Majeti KR, Charlesworth CT, Soupene
            E, Selvaraj S, Jia K, Sheehan VA, Cromer MK, Porteus MH
  TITLE     Enhancement of erythropoietic output by Cas9-mediated insertion of a
            natural variant in haematopoietic stem and progenitor cells.
  JOURNAL   Nat Biomed Eng. 2024 Dec;8(12):1540-1552. doi: 
            10.1038/s41551-024-01222-6. Epub 2024 Jun 17.
  PUBMED    38886504
REFERENCE   2  (bases 1 to 7260)
  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"; doi: 
            "10.1038/s41551-024-01222-6"; journalName: "Nat Biomed Eng"; date: 
            "2024-12"; volume: "8"; issue: "12"; pages: "1540-1552"
COMMENT     Sequence Label: MKC89_CCR5(UbC-tEPOR-2A-YFP)
FEATURES             Location/Qualifiers
     source          1..7260
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             85..138
                     /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"
     CDS             join(139..141,142..144,145..858)
                     /codon_start=1
                     /product="enhanced YFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(184..205)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(445..464)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     792..813
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    859..1083
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   1492..1621
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(1683..2271)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1763..1782)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2442..3233,3234..3302))
                     /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     3065..3084
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3303..3407)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3475..3493
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3531..3553)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3653..3672
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(3689..4144)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3826..3847)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4038..4057
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   4219..4359
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4230..4359
                     /label=AAV2 ITR
     protein_bind    4663..4674
                     /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)"
     promoter        4786..5997
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     5834..5853
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     regulatory      6025..6034
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 gcctcgaagc ccagcccaga gggagcctct gctgccagct ttgagtacac tatcctggac
       61 cccagctccc agctcttgcg tccagagggc aggggcagcc tgctgacctg cggcgacgtg
      121 gaggagaacc ccggccccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
      181 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
      241 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
      301 cccgtgccct ggcccaccct cgtgaccacc ttcggctacg gcctgatgtg cttcgcccgc
      361 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
      421 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
      481 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
      541 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
      601 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
      661 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
      721 ctgctgcccg acaaccacta cctgagctac cagtccaagc tgagcaaaga ccccaacgag
      781 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
      841 gacgagctgt acaagtaact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
      901 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
      961 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     1021 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     1081 tggtgggctc actatgctgc cgcccagtgg gactttggaa atacaatgtg tcaactcttg
     1141 acagggctct attttatagg cttcttctct ggaatcttct tcatcatcct cctgacaatc
     1201 gataggtacc tggctgtcgt ccatgctgtg tttgctttaa aagccaggac ggtcaccttt
     1261 ggggtggtga caagtgtgat cacttgggtg gtggctgtgt ttgcgtctct cccaggaatc
     1321 atctttacca gatctcaaaa agaaggtctt cattacacct gcagctctca ttttccatac
     1381 agtcagtatc aattctggaa gaatttccag acattaaaga tagtcatctt ggggctggtc
     1441 ctgccgctgc ttgtcatggt catctgctac tcgggaatcc taagcggccg caggaacccc
     1501 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac
     1561 caaaggtcgc ccgacgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag
     1621 gacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     1681 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     1741 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     1801 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     1861 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     1921 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     1981 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     2041 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     2101 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     2161 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     2221 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     2281 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     2341 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     2401 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     2461 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     2521 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     2581 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     2641 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     2701 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     2761 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     2821 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     2881 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     2941 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     3001 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     3061 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     3121 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     3181 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     3241 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     3301 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     3361 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     3421 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     3481 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     3541 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     3601 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca
     3661 gagcagattg tactgagagt gcaccataaa attgtaaacg ttaatatttt gttaaaattc
     3721 gcgttaaatt tttgttaaat cagctcattt tttaaccaat agaccgaaat cggcaaaatc
     3781 ccttataaat caaaagaata gcccgagata gagttgagtg ttgttccagt ttggaacaag
     3841 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     3901 gatggcccac tacgtgaacc atcacccaaa tcaagttttt tggggtcgag gtgccgtaaa
     3961 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     4021 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctaaggc gctggcaagt
     4081 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     4141 gcgtactatg gttgctttga cgtatgcggt gtgaaatacc gcacagatgc gtaaggagaa
     4201 aataccgcat caggcgcccc tgcaggcagc tgcgcgctcg ctcgctcact gaggccgccc
     4261 gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc
     4321 gcagagaagg agtggccaac tccatcacta ggggttcctg cggccgcaag agagttaatt
     4381 caatgtagac atctatgtag gcaattaaaa acctattgat gtataaaaca gtttgcattc
     4441 atggagggca actaaataca ttctaggact ttataaaaga tcacttttta tttatgcaca
     4501 gggtggaaca agatggatta tcaagtgtca agtccaatct atgacatcaa ttattataca
     4561 tcggagccct gccaaaaaat caatgtgaag caaatcgcag cccgcctcct gcctccgctc
     4621 tactcactgg tgttcatctt tggttttgtg ggcaacatgc tggtcatcct catcctgata
     4681 aactgcaaaa ggctgaagag catgactgac atctacctgc tcaacctggc catctctgac
     4741 ctgtttttcc ttcttactgt ccccttcatt accctgttat ccctaggcct ccgcgccggg
     4801 ttttggcgcc tcccgcgggc gcccccctcc tcacggcgag cgctgccacg tcagacgaag
     4861 ggcgcagcga gcgtcctgat ccttccgccc ggacgctcag gacagcggcc cgctgctcat
     4921 aagactcggc cttagaaccc cagtatcagc agaaggacat tttaggacgg gacttgggtg
     4981 actctagggc actggttttc tttccagaga gcggaacagg cgaggaaaag tagtcccttc
     5041 tcggcgattc tgcggaggga tctccgtggg gcggtgaacg ccgatgatta tataaggacg
     5101 cgccgggtgt ggcacagcta gttccgtcgc agccgggatt tgggtcgcgg ttcttgtttg
     5161 tggatcgctg tgatcgtcac ttggtgagta gcgggctgct gggctggccg gggctttcgt
     5221 ggccgccggg ccgctcggtg ggacggaagc gtgtggagag accgccaagg gctgtagtct
     5281 gggtccgcga gcaaggttgc cctgaactgg gggttggggg gagcgcagca aaatggcggc
     5341 tgttcccgag tcttgaatgg aagacgcttg tgaggcgggc tgtgaggtcg ttgaaacaag
     5401 gtggggggca tggtgggcgg caagaaccca aggtcttgag gccttcgcta atgcgggaaa
     5461 gctcttattc gggtgagatg ggctggggca ccatctgggg accctgacgt gaagtttgtc
     5521 actgactgga gaactcggtt tgtcgtctgt tgcgggggcg gcagttatgg cggtgccgtt
     5581 gggcagtgca cccgtacctt tgggagcgcg cgccctcgtc gtgtcgtgac gtcacccgtt
     5641 ctgttggctt ataatgcagg gtggggccac ctgccggtag gtgtgcggta ggcttttctc
     5701 cgtcgcagga cgcagggttc gggcctaggg taggctctcc tgaatcgaca ggcgccggac
     5761 ctctggtgag gggagggata agtgaggcgt cagtttcttt ggtcggtttt atgtacctat
     5821 cttcttaagt agctgaagct ccggttttga actatgcgct cggggttggc gagtgtgttt
     5881 tgtgaagttt tttaggcacc ttttgaaatg taatcatttg ggtcaatatg taattttcag
     5941 tgttagacta gtaaattgtc cgctaaattc tggccgtttt tggctttttt gttagacggt
     6001 accgagctct tcgaaggatc catcgccacc atggaccacc tcggggcgtc cctctggccc
     6061 caggtcggct ccctttgtct cctgctcgct ggggccgcct gggcgccccc gcctaacctc
     6121 ccggacccca agttcgagag caaagcggcc ttgctggcgg cccgggggcc cgaagagctt
     6181 ctgtgcttca ccgagcggtt ggaggacttg gtgtgtttct gggaggaagc ggcgagcgct
     6241 ggggtgggcc cgggcaacta cagcttctcc taccagctcg aggatgagcc atggaagctg
     6301 tgtcgcctgc accaggctcc cacggctcgt ggtgcggtgc gcttctggtg ttcgctgcct
     6361 acagccgaca cgtcgagctt cgtgccccta gagttgcgcg tcacagcagc ctccggcgct
     6421 ccgcgatatc accgtgtcat ccacatcaat gaagtagtgc tcctagacgc ccccgtgggg
     6481 ctggtggcgc ggttggctga cgagagcggc cacgtagtgt tgcgctggct cccgccgcct
     6541 gagacaccca tgacgtctca catccgctac gaggtggacg tctcggccgg caacggcgca
     6601 gggagcgtac agagggtgga gatcctggag ggccgcaccg agtgtgtgct gagcaacctg
     6661 cggggccgga cgcgctacac cttcgccgtc cgcgcgcgta tggctgagcc gagcttcggc
     6721 ggcttctgga gcgcctggtc ggagcctgtg tcgctgctga cgcctagcga cctggacccc
     6781 ctcatcctga cgctctccct catcctcgtg gtcatcctgg tgctgctgac cgtgctcgcg
     6841 ctgctctccc accgccgggc tctgaagcag aagatctggc ctggcatccc gagcccagag
     6901 agcgagtttg aaggcctctt caccacccac aagggtaact tccagctgtg gctgtaccag
     6961 aatgatggct gcctgtggtg gagcccctgc acccccttca cggaggaccc acctgcttcc
     7021 ctggaagtcc tctcagagcg ctgctggggg acgatgcagg cagtggagcc ggggacagat
     7081 gatgagggcc ccctgctgga gccagtgggc agtgagcatg cccaggatac ctatctggtg
     7141 ctggacaaat ggttgctgcc ccggaacccg cccagtgagg acctcccagg gcctggtggc
     7201 agtgtggaca tagtggccat ggatgaaggc tcagaagcat cctcctgctc atctgctttg
//
