LOCUS       sequence-465038-        6242 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV production plasmid for SFFV(synEPOR) vector from Figs. 2-3 that 
            mediates HDR at CCR5 locus using CCR5 gRNA. YFP is followed by 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 6242)
  AUTHORS   Shah AP, Majeti KR, Ekman FK, Selvaraj S, Soupene E, Chati P, Sinha 
            R, Luna SE, Charlesworth CT, McCreary T, Lesch BJ, Tran T, Sharma D,
            Chu SN, Porteus MH, Cromer MK
  TITLE     Engineering inducible signaling receptors to enable 
            erythropoietin-free erythropoiesis
  JOURNAL   bioRxiv 2024.04.12.589260
REFERENCE   2  (bases 1 to 6242)
  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: "bioRxiv 
            2024.04.12.589260"
COMMENT     Sequence Label: MKC131_CCR5(SFFV-synEPOR-2A-YFP)
FEATURES             Location/Qualifiers
     source          1..6242
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    28..252
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   661..790
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(852..1440)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(932..951)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1611..2402,2403..2471))
                     /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     2234..2253
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2472..2576)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2644..2662
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2700..2722)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2822..2841
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2858..3313)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2995..3016)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3207..3226
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   3388..3528
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3399..3528
                     /label=AAV2 ITR
     protein_bind    3832..3843
                     /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        4041..4448
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     CDS             4593..4913
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=FKBP-V
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             5496..5549
                     /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(5550..5552,5553..5555,5556..6242,1..27)
                     /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(5595..5616)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5856..5875)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6203..6224
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 ctcggcatgg acgagctgta caagtaactg tgccttctag ttgccagcca tctgttgttt
       61 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
      121 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
      181 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
      241 tgggctctat ggtgggctca ctatgctgcc gcccagtggg actttggaaa tacaatgtgt
      301 caactcttga cagggctcta ttttataggc ttcttctctg gaatcttctt catcatcctc
      361 ctgacaatcg ataggtacct ggctgtcgtc catgctgtgt ttgctttaaa agccaggacg
      421 gtcacctttg gggtggtgac aagtgtgatc acttgggtgg tggctgtgtt tgcgtctctc
      481 ccaggaatca tctttaccag atctcaaaaa gaaggtcttc attacacctg cagctctcat
      541 tttccataca gtcagtatca attctggaag aatttccaga cattaaagat agtcatcttg
      601 gggctggtcc tgccgctgct tgtcatggtc atctgctact cgggaatcct aagcggccgc
      661 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
      721 ccgggcgacc aaaggtcgcc cgacgcccgg gcggcctcag tgagcgagcg agcgcgcagc
      781 tgcctgcagg acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
      841 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
      901 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
      961 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     1021 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     1081 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     1141 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     1201 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     1261 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     1321 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     1381 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     1441 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     1501 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     1561 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     1621 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     1681 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     1741 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     1801 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     1861 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     1921 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     1981 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     2041 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     2101 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     2161 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     2221 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     2281 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     2341 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     2401 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     2461 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     2521 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     2581 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     2641 aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac
     2701 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     2761 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat
     2821 gcggcatcag agcagattgt actgagagtg caccataaaa ttgtaaacgt taatattttg
     2881 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata gaccgaaatc
     2941 ggcaaaatcc cttataaatc aaaagaatag cccgagatag agttgagtgt tgttccagtt
     3001 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     3061 tatcagggcg atggcccact acgtgaacca tcacccaaat caagtttttt ggggtcgagg
     3121 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     3181 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctaaggcg
     3241 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     3301 ctacagggcg cgtactatgg ttgctttgac gtatgcggtg tgaaataccg cacagatgcg
     3361 taaggagaaa ataccgcatc aggcgcccct gcaggcagct gcgcgctcgc tcgctcactg
     3421 aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg
     3481 agcgagcgcg cagagaagga gtggccaact ccatcactag gggttcctgc ggccgcaaga
     3541 gagttaattc aatgtagaca tctatgtagg caattaaaaa cctattgatg tataaaacag
     3601 tttgcattca tggagggcaa ctaaatacat tctaggactt tataaaagat cactttttat
     3661 ttatgcacag ggtggaacaa gatggattat caagtgtcaa gtccaatcta tgacatcaat
     3721 tattatacat cggagccctg ccaaaaaatc aatgtgaagc aaatcgcagc ccgcctcctg
     3781 cctccgctct actcactggt gttcatcttt ggttttgtgg gcaacatgct ggtcatcctc
     3841 atcctgataa actgcaaaag gctgaagagc atgactgaca tctacctgct caacctggcc
     3901 atctctgacc tgtttttcct tcttactgtc cccttcatta ccctgttatc cctaggccga
     3961 taaaataaaa gattttattt agtctccaga aaaagggggg aatgaaagac cccacctgta
     4021 ggtttggcaa gctagctgca gtaacgccat tttgcaaggc atggaaaaat accaaaccaa
     4081 gaatagagaa gttcagatca agggcgggta catgaaaata gctaacgttg ggccaaacag
     4141 gatatctgcg gtgagcagtt tcggccccgg cccggggcca agaacagatg gtcaccgcag
     4201 tttcggcccc ggcccgaggc caagaacaga tggtccccag atatggccca accctcagca
     4261 gtttcttaag acccatcaga tgtttccagg ctcccccaag gacctgaaat gaccctgcgc
     4321 cttatttgaa ttaaccaatc agcctgcttc tcgcttctgt tcgcgcgctt ctgcttcccg
     4381 agctctataa aagagctcac aacccctcac tcggcgcgcc agtcctccga cagactgagt
     4441 cgcccggggg taccgagctc ttcgaaggat ccatcgccac catgaactcc ttctccacaa
     4501 gcgccttcgg tccagttgcc ttctccctgg ggctgctcct ggtgttgcct gctgccttcc
     4561 ctgccccagg cggcggcgga agcatgcttg agggagtgca ggtggaaacc atctccccag
     4621 gagacgggcg caccttcccc aagcgcggcc agacctgcgt ggtgcactac accgggatgc
     4681 ttgaagatgg aaagaaagtt gattcctccc gggacagaaa caagcccttt aagtttatgc
     4741 taggcaagca ggaggtgatc cgaggctggg aagaaggggt tgcccagatg agtgtgggtc
     4801 agagagccaa actgactata tctccagatt atgcctatgg tgccactggg cacccaggca
     4861 tcatcccacc acatgccact ctcgtcttcg atgtggagct tctaaaactg gaaggaggag
     4921 gaggtagtcc cctcatcctg acgctctccc tcatcctcgt ggtcatcctg gtgctgctga
     4981 ccgtgctcgc gctgctctcc caccgccggg ctctgaagca gaagatctgg cctggcatcc
     5041 cgagcccaga gagcgagttt gaaggcctct tcaccaccca caagggtaac ttccagctgt
     5101 ggctgtacca gaatgatggc tgcctgtggt ggagcccctg cacccccttc acggaggacc
     5161 cacctgcttc cctggaagtc ctctcagagc gctgctgggg gacgatgcag gcagtggagc
     5221 cggggacaga tgatgagggc cccctgctgg agccagtggg cagtgagcat gcccaggata
     5281 cctatctggt gctggacaaa tggttgctgc cccggaaccc gcccagtgag gacctcccag
     5341 ggcctggtgg cagtgtggac atagtggcca tggatgaagg ctcagaagca tcctcctgct
     5401 catctgcttt ggcctcgaag cccagcccag agggagcctc tgctgccagc tttgagtaca
     5461 ctatcctgga ccccagctcc cagctcttgc gtccagaggg caggggcagc ctgctgacct
     5521 gcggcgacgt ggaggagaac cccggcccca tggtgagcaa gggcgaggag ctgttcaccg
     5581 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     5641 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     5701 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cttcggctac ggcctgatgt
     5761 gcttcgcccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     5821 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     5881 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     5941 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     6001 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     6061 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     6121 acggccccgt gctgctgccc gacaaccact acctgagcta ccagtccaag ctgagcaaag
     6181 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     6241 ct
//
