LOCUS       sequence-462759-        6789 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV production plasmid for tEPOR-2A-YFP vector from Fig. 2 that 
            mediates HDR at HBA1 locus using HBA1-sg4 gRNA. HBA1 UTRs flank full
            tEPOR-2A-YFP cassette. Homology arms mediate full HBA1 replacement..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6789)
  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 6789)
  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: MKC86_HBA1(tEPOR-2A-YFP)
FEATURES             Location/Qualifiers
     source          1..6789
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(72..91)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     282..303
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(457..476)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     576..598
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(636..654)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        722..826
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(827..895,896..1687)
                     /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(1045..1064)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1858..2446
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2347..2366
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2508..2637
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     CDS             4900..4953
                     /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(4954..4956,4957..4959,4960..5673)
                     /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(4999..5020)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5260..5279)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5607..5628
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     repeat_region   6559..6699
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6559..6688
                     /label=AAV2 ITR
     rep_origin      join(6774..6789,1..440)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
       61 cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
      121 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
      181 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
      241 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
      301 tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat
      361 ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
      421 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata
      481 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
      541 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
      601 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
      661 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
      721 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
      781 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
      841 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
      901 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
      961 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     1021 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     1081 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     1141 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     1201 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     1261 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     1321 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     1381 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     1441 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     1501 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     1561 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     1621 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     1681 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     1741 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     1801 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     1861 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     1921 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     1981 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     2041 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     2101 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     2161 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     2221 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     2281 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     2341 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     2401 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     2461 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgtcct gcaggcagct
     2521 gcgcgctcgc tcgctcactg aggccgcccg ggcgtcgggc gacctttggt cgcccggcct
     2581 cagtgagcga gcgagcgcgc agagagggag tggccaactc catcactagg ggttcctgcg
     2641 ctccagccgg ttccagctat tgctttgttt acctgtttaa ccagtattta cctagcaagt
     2701 cttccatcag atagcatttg gagagctggg ggtgtcacag tgaaccacga cctctaggcc
     2761 agtgggagag tcagtcacac aaactgtgag tccatgactt ggggcttagc ccgcacccac
     2821 caccccacgc gccaccccac aaccccgggt agaggagtct gaatctggag ccgccctcag
     2881 cccagccccc tgctttttgc gtcctggtgt ttattccttc ccggtgcccg tcactcaagc
     2941 acactggtga ctatcgccag agggaaaggg agctgcagga agcgaggctg gagagcagga
     3001 ggggctctgc gcagaaattc ttttgagttc ctatgggcca gggcgtccgg gtgcgcgcat
     3061 tcctctccgc cccaggattg ggcgaagcct cccggctcgc actcgctcgc ccgtgtgttc
     3121 cccgatcccg ctggagtcga tgcgcgtcca gcgcgtgcca ggccggggcg ggggtgcggg
     3181 ctgactttct ccctcgctag ggacgctccg gcgcccgaaa ggaaagggtg gcgctgcgct
     3241 ccggggtgca cgagccgaca gcgcccgacc ccaacgggcc ggccccgcca gcgccgctac
     3301 cgccctgctc ccgggcgagc gggatgggcg ggagtggagt ggcgggtgga gggtggagac
     3361 gtcctggccc ccgccccgcg tgcaccccca ggggaggccg agcccgccgc ccggtcccgc
     3421 gcaggccccg cccgggactc ccttgcggtc caggccgcgc cccgggctcc gcgccagcca
     3481 atgagcgccg cccggccggg cgtgcccccg cgccccaagc ataaaccctg gcgcgctcgc
     3541 ggcccggcac tcttctggtc cccacagact cagagagaac ccaccatgga ccacctcggg
     3601 gcgtccctct ggccccaggt cggctccctt tgtctcctgc tcgctggggc cgcctgggcg
     3661 cccccgccta acctcccgga ccccaagttc gagagcaaag cggccttgct ggcggcccgg
     3721 gggcccgaag agcttctgtg cttcaccgag cggttggagg acttggtgtg tttctgggag
     3781 gaagcggcga gcgctggggt gggcccgggc aactacagct tctcctacca gctcgaggat
     3841 gagccatgga agctgtgtcg cctgcaccag gctcccacgg ctcgtggtgc ggtgcgcttc
     3901 tggtgttcgc tgcctacagc cgacacgtcg agcttcgtgc ccctagagtt gcgcgtcaca
     3961 gcagcctccg gcgctccgcg atatcaccgt gtcatccaca tcaatgaagt agtgctccta
     4021 gacgcccccg tggggctggt ggcgcggttg gctgacgaga gcggccacgt agtgttgcgc
     4081 tggctcccgc cgcctgagac acccatgacg tctcacatcc gctacgaggt ggacgtctcg
     4141 gccggcaacg gcgcagggag cgtacagagg gtggagatcc tggagggccg caccgagtgt
     4201 gtgctgagca acctgcgggg ccggacgcgc tacaccttcg ccgtccgcgc gcgtatggct
     4261 gagccgagct tcggcggctt ctggagcgcc tggtcggagc ctgtgtcgct gctgacgcct
     4321 agcgacctgg accccctcat cctgacgctc tccctcatcc tcgtggtcat cctggtgctg
     4381 ctgaccgtgc tcgcgctgct ctcccaccgc cgggctctga agcagaagat ctggcctggc
     4441 atcccgagcc cagagagcga gtttgaaggc ctcttcacca cccacaaggg taacttccag
     4501 ctgtggctgt accagaatga tggctgcctg tggtggagcc cctgcacccc cttcacggag
     4561 gacccacctg cttccctgga agtcctctca gagcgctgct gggggacgat gcaggcagtg
     4621 gagccgggga cagatgatga gggccccctg ctggagccag tgggcagtga gcatgcccag
     4681 gatacctatc tggtgctgga caaatggttg ctgccccgga acccgcccag tgaggacctc
     4741 ccagggcctg gtggcagtgt ggacatagtg gccatggatg aaggctcaga agcatcctcc
     4801 tgctcatctg ctttggcctc gaagcccagc ccagagggag cctctgctgc cagctttgag
     4861 tacactatcc tggaccccag ctcccagctc ttgcgtccag agggcagggg cagcctgctg
     4921 acctgcggcg acgtggagga gaaccccggc cccatggtga gcaagggcga ggagctgttc
     4981 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     5041 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     5101 accaccggca agctgcccgt gccctggccc accctcgtga ccaccttcgg ctacggcctg
     5161 atgtgcttcg cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     5221 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     5281 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     5341 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     5401 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     5461 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     5521 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gctaccagtc caagctgagc
     5581 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     5641 atcactctcg gcatggacga gctgtacaag taatggccat gcttcttgcc ccttgggcct
     5701 ccccccagcc cctcctcccc ttcctgcacc cgtacccccg tggtctttga ataaagtctg
     5761 agtgggcggc agcctgtgtg tgcctgagtt ttttccctca gcaaacgtgc caggcatggg
     5821 tgtggacagc agctgggaca cacatggcta gaacctctct gcagctggat agggtaggaa
     5881 aaggcagggg cgggaggagg ggatggagga gggaaagtgg agccaccgcg aagtccagct
     5941 ggaaaaacgc tggaccctag agtgctttga ggatgcattt gctctttccc gagttttatt
     6001 cccagacttt tcagattcaa tgcaggtttg ctgaaataat gaatttatcc atctttacgt
     6061 ttctgggcac tcttgtgcca agaactggct ggctttctgc ctgggacgtc actggtttcc
     6121 cagaggtcct cccacatatg ggtggtgggt aggtcagaga agtcccactc cagcatggct
     6181 gcattgatcc cccatcgttc ccactagtct ccgtaaaacc tcccagatac aggcacagtc
     6241 tagatgaaat caggggtgcg gggtgcaact gcaggcccca ggcaattcaa taggggctct
     6301 actttcaccc ccaggtcacc ccagaatgct cacacaccag acactgacgc cctggggctg
     6361 tcaagatcag gcgtttgtct ctgggcccag ctcagggccc agctcagcac ccactcagct
     6421 cccctgaggc tggggagcct gtcccattgc gactggagag gagagcgggg ccacagaggc
     6481 ctggctagaa ggtcccttct ccctggtgtg tgttttctct ctgctgagca ggcttgcagt
     6541 gcctggggta caggccgcag gaacccctag tgatggagtt ggccactcct tctctgcgcg
     6601 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg
     6661 cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc
     6721 tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc
     6781 ctgtagcgg
//
