LOCUS       sequence-462749-        7041 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV production plasmid for tEPOR-UbC-GFP-BGH vector from Fig. 1 that
            mediates HDR at EPOR locus using EPOR-sg1 gRNA. Repair vector 
            introduces premature stop codon into EPOR locus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7041)
  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 7041)
  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: MKC91_tEPOR(UbC-GFP-BGH)
FEATURES             Location/Qualifiers
     source          1..7041
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     946..965
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             1143..1808
                     /codon_start=1
                     /product="green fluorescent protein 2 from Pontellina 
                     plumata, also known as ppluGFP2 (Shagin et al., 2004)"
                     /label=CopGFP
                     /translation="MPAMKIECRITGTLNGVEFELVGGGEGTPEQGRMTNKMKSTKGAL
                     TFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYR
                     YEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDG
                     GYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA
                     "
     CDS             1143..1808
                     /codon_start=1
                     /product="green fluorescent protein 2 from Pontellina 
                     plumata, also known as ppluGFP2 (Shagin et al., 2004)"
                     /label=CopGFP
                     /translation="MPAMKIECRITGTLNGVEFELVGGGEGTPEQGRMTNKMKSTKGAL
                     TFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYR
                     YEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDG
                     GYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA
                     "
     repeat_region   2794..2934
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2794..2923
                     /label=AAV2 ITR
     rep_origin      3009..3464
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3096..3115)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3306..3327
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3481..3500)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3600..3622
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3660..3678)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3746..3850
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3851..3919,3920..4711)
                     /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(4069..4088)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4882..5470
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5371..5390
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5532..5661
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     primer_bind     complement(6661..6678)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6667..6891
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        join(6939..7041,1..1109)
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
ORIGIN
        1 ccggacgctc aggacagcgg cccgctgctc ataagactcg gccttagaac cccagtatca
       61 gcagaaggac attttaggac gggacttggg tgactctagg gcactggttt tctttccaga
      121 gagcggaaca ggcgaggaaa agtagtccct tctcggcgat tctgcggagg gatctccgtg
      181 gggcggtgaa cgccgatgat tatataagga cgcgccgggt gtggcacagc tagttccgtc
      241 gcagccggga tttgggtcgc ggttcttgtt tgtggatcgc tgtgatcgtc acttggtgag
      301 tagcgggctg ctgggctggc cggggctttc gtggccgccg ggccgctcgg tgggacggaa
      361 gcgtgtggag agaccgccaa gggctgtagt ctgggtccgc gagcaaggtt gccctgaact
      421 gggggttggg gggagcgcag caaaatggcg gctgttcccg agtcttgaat ggaagacgct
      481 tgtgaggcgg gctgtgaggt cgttgaaaca aggtgggggg catggtgggc ggcaagaacc
      541 caaggtcttg aggccttcgc taatgcggga aagctcttat tcgggtgaga tgggctgggg
      601 caccatctgg ggaccctgac gtgaagtttg tcactgactg gagaactcgg tttgtcgtct
      661 gttgcggggg cggcagttat ggcggtgccg ttgggcagtg cacccgtacc tttgggagcg
      721 cgcgccctcg tcgtgtcgtg acgtcacccg ttctgttggc ttataatgca gggtggggcc
      781 acctgccggt aggtgtgcgg taggcttttc tccgtcgcag gacgcagggt tcgggcctag
      841 ggtaggctct cctgaatcga caggcgccgg acctctggtg aggggaggga taagtgaggc
      901 gtcagtttct ttggtcggtt ttatgtacct atcttcttaa gtagctgaag ctccggtttt
      961 gaactatgcg ctcggggttg gcgagtgtgt tttgtgaagt tttttaggca ccttttgaaa
     1021 tgtaatcatt tgggtcaata tgtaattttc agtgttagac tagtaaattg tccgctaaat
     1081 tctggccgtt tttggctttt ttgttagacg gtaccgagct cttcgaagga tccatcgcca
     1141 ccatgcccgc catgaagatc gagtgccgca tcaccggcac cctgaacggc gtggagttcg
     1201 agctggtggg cggcggagag ggcacccccg agcagggccg catgaccaac aagatgaaga
     1261 gcaccaaagg cgccctgacc ttcagcccct acctgctgag ccacgtgatg ggctacggct
     1321 tctaccactt cggcacctac cccagcggct acgagaaccc cttcctgcac gccatcaaca
     1381 acggcggcta caccaacacc cgcatcgaga agtacgagga cggcggcgtg ctgcacgtga
     1441 gcttcagcta ccgctacgag gccggccgcg tgatcggcga cttcaaggtg gtgggcaccg
     1501 gcttccccga ggacagcgtg atcttcaccg acaagatcat ccgcagcaac gccaccgtgg
     1561 agcacctgca ccccatgggc gataacgtgc tggtgggcag cttcgcccgc accttcagcc
     1621 tgcgcgacgg cggctactac agcttcgtgg tggacagcca catgcacttc aagagcgcca
     1681 tccaccccag catcctgcag aacgggggcc ccatgttcgc cttccgccgc gtggaggagc
     1741 tgcacagcaa caccgagctg ggcatcgtgg agtaccagca cgccttcaag acccccatcg
     1801 ccttcgccag atctcgagtc tagacactgt gccctgagct gccccctacc ccaccccacc
     1861 taaagtacct gtaccttgtg gtatctgact ctggcatctc aactgactac agctcagggg
     1921 actcccaggg agcccaaggg ggcttatccg atggccccta ctccaaccct tatgagaaca
     1981 gccttatccc agccgctgag cctctgcccc ccagctatgt ggcttgctct taggacacca
     2041 ggctgcagat gatcagggat ccaatatgac tcagagaacc agtgcagact caagacttat
     2101 ggaacaggga tggcgaggcc tctctcagga gcaggggcat tgctgatttt gtctgcccaa
     2161 tccatcctgc tcaggaaacc acaaccttgc agtattttta aatatgtata gttttttttt
     2221 gtatctatat atatatatac acatatgtat gtaagttttt ctaccatgat ttctacaaac
     2281 accctttaag tcccatcttc ccctgggcat aggccatagg gatagaagtt aaagttcttg
     2341 agcttattca gaagctggat ctgcaatctg aatgctactc ataacataac aaaatagtat
     2401 gttaaacagc tcttaaatct tactggctta ccacattaaa tgatttctct ctcctaactc
     2461 agctcaaatg ggcagccatc catgggatga gtcagaggtt cagactcttc cagtctgtag
     2521 ctctaccttc tcttagggta cttagatgga tcccctgttc tacaaactgc cagtcagcaa
     2581 gggaagaaaa agggcagcaa tgaccctcaa tgggccattt gagggatctg gcctggaaat
     2641 gggcttcctc tcttcttctc acacctcact ggctggaaac agtcacatga ccccagtcac
     2701 atgaaaggcc aggaaactta gtttagctgt acacccagga agggcaaagc tgtttaaggg
     2761 ccactagcta gtctctgcca ctaatgcggc cgcaggaacc cctagtgatg gagttggcca
     2821 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     2881 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc
     2941 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca
     3001 accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3061 cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt
     3121 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     3181 ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg
     3241 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     3301 taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt
     3361 tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca
     3421 aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag
     3481 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     3541 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     3601 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     3661 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     3721 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     3781 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     3841 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     3901 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     3961 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     4021 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     4081 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     4141 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     4201 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     4261 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     4321 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     4381 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     4441 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     4501 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     4561 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     4621 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     4681 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     4741 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     4801 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     4861 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     4921 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     4981 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     5041 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     5101 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     5161 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     5221 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     5281 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     5341 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     5401 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     5461 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     5521 tgctcacatg tcctgcaggc agctgcgcgc tcgctcgctc actgaggccg cccgggcgtc
     5581 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
     5641 actccatcac taggggttcc tgcggccgcg agatgggggt ctcactatgt ttcctaggct
     5701 ggtcttgaac tcctgggttc aaatgatcct cccacctcag cctcccaaag tactgggatt
     5761 atagtgctgg tgtaaaccac tgcacctggc catggccagg attaaaggga gaatgaccaa
     5821 ggtatattga actcctatgc acccttcaat accctgttcc atttaccctt ttgtagggcc
     5881 ttgctgatgc ttcagccaaa acccctgtcc cctggccctg atgtactcct ctgcctccat
     5941 tgtgatcaca gggaccaagt gtatctgtgc ctctatgact gggagtggag ggggaattgg
     6001 tgagtattca atgagtcata tctatgtaac tatttatatt ggcttcaaca gggctctgaa
     6061 gcagaagatc tggcctggca tcccgagccc agagagcgag tttgaaggcc tcttcaccac
     6121 ccacaagggt aacttccagg taggtggcct ggttgtcccc tcagtgcctg ggcttccctg
     6181 cttcttgcag ccaaactgca ggcctctctg agcaggttgg tgctatttct tcagctgtgg
     6241 ctgtaccaga atgatggctg cctgtggtgg agcccctgca cccccttcac ggaggaccca
     6301 cctgcttccc tggaagtcct ctcagagcgc tgctggggga cgatgcaggc agtggagccg
     6361 gggacagatg atgagggccc cctgctggag ccagtgggca gtgagcatgc ccaggatacc
     6421 tatctggtgc tggacaaatg gttgctgccc cggaacccgc ccagtgagga cctcccaggg
     6481 cctggtggca gtgtggacat agtggccatg gatgaaggct cagaagcatc ctcctgctca
     6541 tctgctttgg cctcgaagcc cagcccagag ggagcctctg ctgccagctt tgagtacact
     6601 atcctggacc ccagctccca gctcttgcgt ccatagctcg agggcgcgcc cgctgatcag
     6661 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     6721 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     6781 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     6841 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     6901 cggaaagaac gtttcgcgcc attaccctgt tatccctagg cctccgcgcc gggttttggc
     6961 gcctcccgcg ggcgcccccc tcctcacggc gagcgctgcc acgtcagacg aagggcgcag
     7021 cgagcgtcct gatccttccg c
//
