LOCUS       sequence-173848-        7412 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-terminally EGFP-tagged human HIF-2a/EPAS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7412)
  AUTHORS   Bagnall J, Leedale J, Taylor SE, Spiller DG, White MR, Sharkey KJ, 
            Bearon RN, See V
  TITLE     Tight control of hypoxia-inducible factor-alpha transient dynamics 
            is essential for cell survival in hypoxia.
  JOURNAL   J Biol Chem. 2014 Feb 28;289(9):5549-64. doi: 
            10.1074/jbc.M113.500405. Epub 2014 Jan 6.
  PUBMED    24394419
REFERENCE   2  (bases 1 to 7412)
  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: 
            "10.1074/jbc.M113.500405"; journalName: "J Biol Chem"; date: 
            "2014-02-28- 28"; volume: "289"; issue: "9"; pages: "5549-64"
COMMENT     Sequence Label: pg-EGFP-HIF-2alpha
FEATURES             Location/Qualifiers
     source          1..7412
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      547..556
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(553..555,556..558,559..1269)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(553..555,556..558,559..1269)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(598..619)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(859..878)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1206..1227
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    1297..1321
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3941..3965)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    4140..4261
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4177..4196)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4231..4250
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4268..4723)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4405..4426)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4617..4636
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4750..4854
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4856..5213
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4856..5047
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4879..4899)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5064..5199
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5248..6042
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             5248..6042
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5302..5321)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5912..5931
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6230..6249)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6274..6321
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6650..7238
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7139..7158
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      541 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      601 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      661 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      721 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      781 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      841 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      901 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
      961 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1021 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1081 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1141 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1201 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1261 ctgtacaagt ccggccggac tcagatctcg agatcaacaa gtttgtacaa aaaagcaggc
     1321 ttcaccatga cagctgacaa ggagaagaaa aggagtagct cggagaggag gaaggagaag
     1381 tcccgggatg ctgcgcggtg ccggcggagc aaggagacgg aggtgttcta tgagctggcc
     1441 catgagctgc ctctgcccca cagtgtgagc tcccatctgg acaaggcctc catcatgcga
     1501 ctggcaatca gcttcctgcg aacacacaag ctcctctcct cagtttgctc tgaaaacgag
     1561 tccgaagccg aagctgacca gcagatggac aacttgtacc tgaaagcctt ggagggtttc
     1621 attgccgtgg tgacccaaga tggcgacatg atctttctgt cagaaaacat cagcaagttc
     1681 atgggactta cacaggtgga gctaacagga catagtatct ttgacttcac tcatccctgc
     1741 gaccatgagg agattcgtga gaacctgagt ctcaaaaatg gctctggttt tgggaaaaaa
     1801 agcaaagaca tgtccacaga gcgggacttc ttcatgagga tgaagtgcac ggtcaccaac
     1861 agaggccgta ctgtcaacct caagtcagcc acctggaagg tcttgcactg cacgggccag
     1921 gtgaaagtct acaacaactg ccctcctcac aatagtctgt gtggctacaa ggagcccctg
     1981 ctgtcctgcc tcatcatcat gtgtgaacca atccagcacc catcccacat ggacatcccc
     2041 ctggatagca agaccttcct gagccgccac agcatggaca tgaagttcac ctactgtgat
     2101 gacagaatca cagaactgat tggttaccac cctgaggagc tgcttggccg ctcagcctat
     2161 gaattctacc atgcgctaga ctccgagaac atgaccaaga gtcaccagaa cttgtgcacc
     2221 aagggtcagg tagtaagtgg ccagtaccgg atgctcgcaa agcatggggg ctacgtgtgg
     2281 ctggagaccc aggggacggt catctacaac cctcgcaacc tgcagcccca gtgcatcatg
     2341 tgtgtcaact acgtcctgag tgagattgag aagaatgacg tggtgttctc catggaccag
     2401 actgaatccc tgttcaagcc ccacctgatg gccatgaaca gcatctttga tagcagtggc
     2461 aagggggctg tgtctgagaa gagtaacttc ctattcacca agctaaagga ggagcccgag
     2521 gagctggccc agctggctcc caccccagga gacgccatca tctctctgga tttcgggaat
     2581 cagaacttcg aggagtcctc agcctatggc aaggccatcc tgcccccgag ccagccatgg
     2641 gccacggagt tgaggagcca cagcacccag agcgaggctg ggagcctgcc tgccttcacc
     2701 gtgccccagg cagctgcccc gggcagcacc acccccagtg ccaccagcag cagcagcagc
     2761 tgctccacgc ccaatagccc tgaagactat tacacatctt tggataacga cctgaagatt
     2821 gaagtgattg agaagctctt cgccatggac acagaggcca aggaccaatg cagtacccag
     2881 acggatttca atgagctgga cttggagaca ctggcaccct atatccccat ggacggggaa
     2941 gacttccagc taagccccat ctgccccgag gagcggctct tggcggagaa cccacagtcc
     3001 accccccagc actgcttcag tgccatgaca aacatcttcc agccactggc ccctgtagcc
     3061 ccgcacagtc ccttcctcct ggacaagttt cagcagcagc tggagagcaa gaagacagag
     3121 cccgagcacc ggcccatgtc ctccatcttc tttgatgccg gaagcaaagc atccctgcca
     3181 ccgtgctgtg gccaggccag cacccctctc tcttccatgg ggggcagatc caatacccag
     3241 tggcccccag atccaccatt acattttggg cccacaaagt gggccgtcgg ggatcagcgc
     3301 acagagttct tgggagcagc gccgttgggg ccccctgtct ctccacccca tgtctccacc
     3361 ttcaagacaa ggtctgcaaa gggttttggg gctcgaggcc cagacgtgct gagtccggcc
     3421 atggtagccc tctccaacaa gctgaagctg aagcgacagc tggagtatga agagcaagcc
     3481 ttccaggacc tgagcggggg ggacccacct ggtggcagca cctcacattt gatgtggaaa
     3541 cggatgaaga acctcagggg tgggagctgc cctttgatgc cggacaagcc actgagcgca
     3601 aatgtaccca atgataagtt cacccaaaac cccatgaggg gcctgggcca tcccctgaga
     3661 catctgccgc tgccacagcc tccatctgcc atcagtcccg gggagaacag caagagcagg
     3721 ttccccccac agtgctacgc cacccagtac caggactaca gcctgtcgtc agcccacaag
     3781 gtgtcaggca tggcaagccg gctgctcggg ccctcatttg agtcctacct gctgcccgaa
     3841 ctgaccagat atgactgtga ggtgaacgtg cccgtgctgg gaagctccac gctcctgcaa
     3901 ggaggggacc tcctcagagc cctggacgag gccacctgag acccagcttt cttgtacaaa
     3961 gtggttgatc tcaagcttcg aattctgcag tcgacggtac cgcgggcccg ggatccaccg
     4021 gatctagata actgatcata atcagccata ccacatttgt agaggtttta cttgctttaa
     4081 aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta
     4141 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     4201 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     4261 aacgcgtaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     4321 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     4381 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     4441 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     4501 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     4561 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     4621 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     4681 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg
     4741 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     4801 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtcctgag
     4861 gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     4921 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     4981 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     5041 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     5101 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     5161 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaagatcgat
     5221 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     5281 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     5341 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     5401 gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc gtggctggcc
     5461 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     5521 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     5581 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     5641 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     5701 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     5761 gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     5821 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     5881 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     5941 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     6001 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     6061 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     6121 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     6181 gcggggatct catgctggag ttcttcgccc accctagggg gaggctaact gaaacacgga
     6241 aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag aataaaacgc
     6301 acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg cactctgtcg
     6361 ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt tccccacccc
     6421 accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg
     6481 ccatagcctc aggttactca tatatacttt agattgattt aaaacttcat ttttaattta
     6541 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     6601 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     6661 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     6721 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     6781 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     6841 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     6901 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     6961 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     7021 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     7081 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     7141 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     7201 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     7261 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     7321 attctgtgga taaccgtatt accgccatgc attagttatt aatagtaatc aattacgggg
     7381 tcattagttc atagcccata tatggagttc cg
//
