LOCUS       sequence-170214-        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"
     polyA_signal    189..310
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(226..245)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     280..299
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(317..772)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(454..475)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     666..685
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        799..903
                     /gene="bla"
                     /label=AmpR promoter
     promoter        905..1262
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        905..1096
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(928..948)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      1113..1248
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             1297..2091
                     /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             1297..2091
                     /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(1351..1370)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1961..1980
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(2279..2298)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2323..2370
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2699..3287
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3188..3207
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        3462..3765
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3766..3969
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3920..3940
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      4008..4017
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4014..4016,4017..4019,4020..4730)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4014..4016,4017..4019,4020..4730)
                     /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(4059..4080)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4320..4339)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4667..4688
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    4758..4782
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(join(7402..7412,1..14))
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 ttgtacaaag tggttgatct caagcttcga attctgcagt cgacggtacc gcgggcccgg
       61 gatccaccgg atctagataa ctgatcataa tcagccatac cacatttgta gaggttttac
      121 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
      181 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
      241 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
      301 atgtatctta acgcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
      361 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
      421 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
      481 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
      541 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
      601 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
      661 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
      721 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc
      781 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
      841 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
      901 agtcctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc
      961 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     1021 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     1081 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     1141 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     1201 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     1261 aagatcgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     1321 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     1381 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     1441 gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg
     1501 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     1561 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     1621 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     1681 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     1741 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     1801 gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat
     1861 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     1921 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     1981 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     2041 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     2101 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     2161 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     2221 tcctccagcg cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg
     2281 aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga
     2341 ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc
     2401 actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt
     2461 ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg
     2521 caggccctgc catagcctca ggttactcat atatacttta gattgattta aaacttcatt
     2581 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     2641 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     2701 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     2761 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     2821 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     2881 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     2941 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     3001 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     3061 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     3121 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     3181 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     3241 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     3301 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     3361 tatcccctga ttctgtggat aaccgtatta ccgccatgca ttagttatta atagtaatca
     3421 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     3481 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     3541 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     3601 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     3661 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     3721 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
     3781 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
     3841 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
     3901 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
     3961 agcagagctg gtttagtgaa ccgtcagatc cgctagcgct accggtcgcc accatggtga
     4021 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     4081 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     4141 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     4201 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
     4261 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     4321 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     4381 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     4441 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
     4501 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     4561 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     4621 gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     4681 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag tccggccgga
     4741 ctcagatctc gagatcaaca agtttgtaca aaaaagcagg cttcaccatg acagctgaca
     4801 aggagaagaa aaggagtagc tcggagagga ggaaggagaa gtcccgggat gctgcgcggt
     4861 gccggcggag caaggagacg gaggtgttct atgagctggc ccatgagctg cctctgcccc
     4921 acagtgtgag ctcccatctg gacaaggcct ccatcatgcg actggcaatc agcttcctgc
     4981 gaacacacaa gctcctctcc tcagtttgct ctgaaaacga gtccgaagcc gaagctgacc
     5041 agcagatgga caacttgtac ctgaaagcct tggagggttt cattgccgtg gtgacccaag
     5101 atggcgacat gatctttctg tcagaaaaca tcagcaagtt catgggactt acacaggtgg
     5161 agctaacagg acatagtatc tttgacttca ctcatccctg cgaccatgag gagattcgtg
     5221 agaacctgag tctcaaaaat ggctctggtt ttgggaaaaa aagcaaagac atgtccacag
     5281 agcgggactt cttcatgagg atgaagtgca cggtcaccaa cagaggccgt actgtcaacc
     5341 tcaagtcagc cacctggaag gtcttgcact gcacgggcca ggtgaaagtc tacaacaact
     5401 gccctcctca caatagtctg tgtggctaca aggagcccct gctgtcctgc ctcatcatca
     5461 tgtgtgaacc aatccagcac ccatcccaca tggacatccc cctggatagc aagaccttcc
     5521 tgagccgcca cagcatggac atgaagttca cctactgtga tgacagaatc acagaactga
     5581 ttggttacca ccctgaggag ctgcttggcc gctcagccta tgaattctac catgcgctag
     5641 actccgagaa catgaccaag agtcaccaga acttgtgcac caagggtcag gtagtaagtg
     5701 gccagtaccg gatgctcgca aagcatgggg gctacgtgtg gctggagacc caggggacgg
     5761 tcatctacaa ccctcgcaac ctgcagcccc agtgcatcat gtgtgtcaac tacgtcctga
     5821 gtgagattga gaagaatgac gtggtgttct ccatggacca gactgaatcc ctgttcaagc
     5881 cccacctgat ggccatgaac agcatctttg atagcagtgg caagggggct gtgtctgaga
     5941 agagtaactt cctattcacc aagctaaagg aggagcccga ggagctggcc cagctggctc
     6001 ccaccccagg agacgccatc atctctctgg atttcgggaa tcagaacttc gaggagtcct
     6061 cagcctatgg caaggccatc ctgcccccga gccagccatg ggccacggag ttgaggagcc
     6121 acagcaccca gagcgaggct gggagcctgc ctgccttcac cgtgccccag gcagctgccc
     6181 cgggcagcac cacccccagt gccaccagca gcagcagcag ctgctccacg cccaatagcc
     6241 ctgaagacta ttacacatct ttggataacg acctgaagat tgaagtgatt gagaagctct
     6301 tcgccatgga cacagaggcc aaggaccaat gcagtaccca gacggatttc aatgagctgg
     6361 acttggagac actggcaccc tatatcccca tggacgggga agacttccag ctaagcccca
     6421 tctgccccga ggagcggctc ttggcggaga acccacagtc caccccccag cactgcttca
     6481 gtgccatgac aaacatcttc cagccactgg cccctgtagc cccgcacagt cccttcctcc
     6541 tggacaagtt tcagcagcag ctggagagca agaagacaga gcccgagcac cggcccatgt
     6601 cctccatctt ctttgatgcc ggaagcaaag catccctgcc accgtgctgt ggccaggcca
     6661 gcacccctct ctcttccatg gggggcagat ccaataccca gtggccccca gatccaccat
     6721 tacattttgg gcccacaaag tgggccgtcg gggatcagcg cacagagttc ttgggagcag
     6781 cgccgttggg gccccctgtc tctccacccc atgtctccac cttcaagaca aggtctgcaa
     6841 agggttttgg ggctcgaggc ccagacgtgc tgagtccggc catggtagcc ctctccaaca
     6901 agctgaagct gaagcgacag ctggagtatg aagagcaagc cttccaggac ctgagcgggg
     6961 gggacccacc tggtggcagc acctcacatt tgatgtggaa acggatgaag aacctcaggg
     7021 gtgggagctg ccctttgatg ccggacaagc cactgagcgc aaatgtaccc aatgataagt
     7081 tcacccaaaa ccccatgagg ggcctgggcc atcccctgag acatctgccg ctgccacagc
     7141 ctccatctgc catcagtccc ggggagaaca gcaagagcag gttcccccca cagtgctacg
     7201 ccacccagta ccaggactac agcctgtcgt cagcccacaa ggtgtcaggc atggcaagcc
     7261 ggctgctcgg gccctcattt gagtcctacc tgctgcccga actgaccaga tatgactgtg
     7321 aggtgaacgt gcccgtgctg ggaagctcca cgctcctgca aggaggggac ctcctcagag
     7381 ccctggacca ggccacctga gacccagctt tc
//
