LOCUS       sequence-465998-        7204 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express mouse homeostatic iron regulator (HFE)(H67D) from an EF1a
            promoter. It also encodes GFP after an IRES sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7204)
  TITLE     Ploski Lab Plasmids 2025
REFERENCE   2  (bases 1 to 7204)
  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"
COMMENT     Sequence Label: pAAV-EF1a-mHFE(H67D)-IRES-GFP-WPRE-pA
FEATURES             Location/Qualifiers
     source          1..7204
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   651..780
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        795..1973
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1026..1964
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1921..1941
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     misc_feature    3093..3666
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3261..3278)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3488..3507
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(3669..3671,3672..3674,3675..4388)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3669..3671,3672..3674,3675..4385)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3714..3735)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3975..3994)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4322..4343
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    4456..5044
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4509..4529)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4927..4938)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    5052..5100
                     /note="synthetic polyadenylation signal"
     repeat_region   5117..5257
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5117..5246
                     /label=AAV2 ITR
     rep_origin      5332..5787
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5419..5438)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5629..5650
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5804..5823)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5923..5945
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5983..6001)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6069..6173
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6174..6242,6243..7034)
                     /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(6392..6411)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca
      661 gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg
      721 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
      781 gcggccgcac gcgtggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc
      841 ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg
      901 taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac
      961 cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa
     1021 cacaggtaag tgccgtgtgt ggttcccgcg ggcctggcct ctttacgggt tatggccctt
     1081 gcgtgccttg aattacttcc acctggctgc agtacgtgat tcttgatccc gagcttcggg
     1141 ttggaagtgg gtgggagagt tcgaggcctt gcgcttaagg agccccttcg cctcgtgctt
     1201 gagttgaggc ctggcctggg cgctggggcc gccgcgtgcg aatctggtgg caccttcgcg
     1261 cctgtctcgc tgctttcgat aagtctctag ccatttaaaa tttttgatga cctgctgcga
     1321 cgcttttttt ctggcaagat agtcttgtaa atgcgggcca agatctgcac actggtattt
     1381 cggtttttgg ggccgcgggc ggcgacgggg cccgtgcgtc ccagcgcaca tgttcggcga
     1441 ggcggggcct gcgagcgcgg ccaccgagaa tcggacgggg gtagtctcaa gctggccggc
     1501 ctgctctggt gcctggcctc gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg
     1561 cccggtcggc accagttgcg tgagcggaaa gatggccgct tcccggccct gctgcaggga
     1621 gctcaaaatg gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc acacaaagga
     1681 aaagggcctt tccgtcctca gccgtcgctt catgtgactc cacggagtac cgggcgccgt
     1741 ccaggcacct cgattagttc tcgagctttt ggagtacgtc gtctttaggt tggggggagg
     1801 ggttttatgc gatggagttt ccccacactg agtgggtgga gactgaagtt aggccagctt
     1861 ggcacttgat gtaattctcc ttggaatttg ccctttttga gtttggatct tggttcattc
     1921 tcaagcctca gacagtggtt caaagttttt ttcttccatt tcaggtgtcg tgaatcgatg
     1981 ccaccatgag cctatcagct gggctccctg tgcggccgct gctgctgctg ctgctactgc
     2041 tgtggtccgt ggccccgcag gcactgccac cgcgttcaca ttctctaaga tacctcttca
     2101 tgggtgcctc agagccagac ctcgggctgc ctttgtttga ggctaggggc tatgtggatg
     2161 accagctctt tgtgtcctac aatgatgaga gtcgccgtgc tgagcccagg gccccgtgga
     2221 tcttggagca aacctcaagc cagctgtggc tgcatctgag tcagagcctg aaagggtggg
     2281 actacatgtt catagtagac ttctggacca tcatgggcaa ctataaccac agtaaggtca
     2341 cgaagttggg agtggtgtcc gagtcccaca tcctgcaggt ggtcctaggc tgtgaggtgc
     2401 atgaagacaa cagtaccagc ggcttctgga gatatggtta tgacgggcaa gatcacctgg
     2461 aattctgccc caagacacta aactggagcg cagccgagcc aggggcctgg gccaccaagg
     2521 tggaatggga cgagcacaag atccgtgcca aacagaacag ggactacctg gagaaggact
     2581 gccccgagca gctgaaacgg ctcctggagc tggggagagg cgttctggga cagcaagtgc
     2641 ctactttggt gaaagtgact cgccactggg cctctacggg gacctctcta aggtgtcagg
     2701 ctctagactt cttcccccag aacatcacta tgaggtggtt gaaggacaac caaccactgg
     2761 atgccaaaga tgtcaacccc gagaaggtgc tacctaacgg ggatgagacc tatcaaggct
     2821 ggctgacatt ggccgtggcc cctggggacg agacaaggtt cacctgtcaa gtggagcacc
     2881 caggcctgga ccagcctctc actgcctctt gggagccctt gcaatctcag gccatgatta
     2941 ttggaatcat cagtggggtc accatctgtg ccatcttctt agttggaatt ctgttcctaa
     3001 tcttaaggaa aaggaaggct tcaggaggaa ccatgggtgg ctatgtctta acagactgtg
     3061 agtgacccgg gctgcaggaa ttcgatggcc gccccctctc cctccccccc cccctaacgt
     3121 tactggccga agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac
     3181 catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag
     3241 cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa
     3301 ggaagcagtt cctctggaag cttcttgaag acaaacaacg tctgtagcga ccctttgcag
     3361 gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga
     3421 tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag
     3481 agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc
     3541 ccattgtatg ggatctgatc tggggcctcg gtgcacatgc tttacatgtg tttagtcgag
     3601 gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg gtttcccttt gaaaaacacg
     3661 ataataccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     3721 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     3781 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     3841 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     3901 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     3961 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     4021 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     4081 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     4141 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     4201 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     4261 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
     4321 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     4381 acaagtaatc cggactcaga tccagcgctg ataacccagc ctccaccaca aacaaggatg
     4441 ccggtaccgg ccgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     4501 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     4561 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct
     4621 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct
     4681 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc
     4741 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg
     4801 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc
     4861 tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac
     4921 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg
     4981 cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc
     5041 ccgcggaatt caataaaaga tctttatttt cattagatct gtgtgttggt tttttgtgtg
     5101 cggaccgagc ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     5161 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
     5221 gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc
     5281 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
     5341 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     5401 ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     5461 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     5521 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
     5581 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     5641 tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta taagggattt
     5701 tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     5761 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
     5821 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     5881 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     5941 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     6001 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     6061 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     6121 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     6181 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     6241 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     6301 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     6361 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     6421 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     6481 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     6541 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     6601 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     6661 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     6721 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     6781 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     6841 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     6901 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     6961 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     7021 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     7081 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     7141 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     7201 cttc
//
