LOCUS       sequence-173844-        7275 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-terminally EGFP-tagged human HIF-1a.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7275)
  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 7275)
  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-HIF-1alpha-EGFP
FEATURES             Location/Qualifiers
     source          1..7275
                     /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"
     protein_bind    563..587
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3072..3096)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      3155..3164
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3161..3163,3164..3166,3167..3880)
                     /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(3161..3163,3164..3166,3167..3877)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3206..3227)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3467..3486)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3814..3835
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4003..4124
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4040..4059)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4094..4113
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4131..4586)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4268..4289)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4480..4499
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4613..4717
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4719..5076
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4719..4910
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4742..4762)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4927..5062
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5111..5905
                     /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             5111..5905
                     /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(5165..5184)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5775..5794
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6093..6112)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6137..6184
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6513..7101
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7002..7021
                     /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 ccggactcag atctcgagat caacaagttt gtacaaaaaa gcaggcttca ccatggaggg
      601 cgccggcggc gcgaacgaca agaaaaagat aagttctgaa cgtcgaaaag aaaagtctcg
      661 agatgcagcc agatctcggc gaagtaaaga atctgaagtt ttttatgagt ttgctcatca
      721 gttgccactt ccacataatg tgagttcgca tcttgataag gcctctgtga tgaggcttac
      781 catcagctat ttgcgtgtga ggaaacttct ggatgctggt gatttggata ttgaagatga
      841 catgaaagca caggtgaatt gcttttattt gaaagccttg gatggttttg ttatggttct
      901 cacagatgat ggtgacatga tttacatttc tgataatgtg aacaaataca tgggattaac
      961 tcagtttgaa ctaactggac acagtgtgtt tgattttact catccatgtg accatgagga
     1021 aatgagagaa atgcttacac acagaaatgg ccttgtgaaa aagggtaaag aacaaaacac
     1081 acagcgaagc ttttttctca gaatgaagtg taccctaact agccgaggaa gaactatgaa
     1141 cataaagtct gcaacatgga aggtattgca ctgcacaggc cacattcacg tatatgatac
     1201 caacagtaac caacctcagt gtgggtataa gaaaccacct atgacctgct tggtgctgat
     1261 ttgtgaaccc attcctcacc catcaaatat tgaaattcct ttagatagca agactttcct
     1321 cagtcgacac agcctggata tgaaattttc ttattgtgat gaaagaatta ccgaattgat
     1381 gggatatgag ccagaagaac ttttaggccg ctcaatttat gaatattatc atgctttgga
     1441 ctctgatcat ctgaccaaaa ctcatcatga tatgtttact aaaggacaag tcaccacagg
     1501 acagtacagg atgcttgcca aaagaggtgg atatgtctgg gttgaaactc aagcaactgt
     1561 catatataac accaagaatt ctcaaccaca gtgcattgta tgtgtgaatt acgttgtgag
     1621 tggtattatt cagcacgact tgattttctc ccttcaacaa acagaatgtg tccttaaacc
     1681 ggttgaatct tcagatatga aaatgactca gctattcacc aaagttgaat cagaagatac
     1741 aagtagcctc tttgacaaac ttaagaagga acctgatgct ttaactttgc tggccccagc
     1801 cgctggagac acaatcatat ctttagattt tggcagcaac gacacagaaa ctgatgacca
     1861 gcaacttgag gaagtaccat tatataatga tgtaatgctc ccctcaccca acgaaaaatt
     1921 acagaatata aatttggcaa tgtctccatt acccaccgct gaaacgccaa agccacttcg
     1981 aagtagtgct gaccctgcac tcaatcaaga agttgcatta aaattagaac caaatccaga
     2041 gtcactggaa ctttctttta ccatgcccca gattcaggat cagacaccta gtccttccga
     2101 tggaagcact agacaaagtt cacctgagcc taatagtccc agtgaatatt gtttttatgt
     2161 ggatagtgat atggtcaatg aattcaagtt ggaattggta gaaaaacttt ttgctgaaga
     2221 cacagaagca aagaacccat tttctactca ggacacagat ttagacttgg agatgttagc
     2281 tccctatatc ccaatggatg atgacttcca gttacgttcc ttcgatcagt tgtcaccatt
     2341 agaaagcagt tccgcaagcc ctgaaagcgc aagtcctcaa agcacagtta cagtattcca
     2401 gcagactcaa atacaagaac ctactgctaa tgccaccact accactgcca ccactgatga
     2461 attaaaaaca gtgacaaaag accgtatgga agacattaaa atattgattg catctccatc
     2521 tcctacccac atacataaag aaactactag tgccacatca tcaccatata gagatactca
     2581 aagtcggaca gcctcaccaa acagagcagg aaaaggagtc atagaacaga cagaaaaatc
     2641 tcatccaaga agccctaacg tgttatctgt cgctttgagt caaagaacta cagttcctga
     2701 ggaagaacta aatccaaaga tactagcttt gcagaatgct cagagaaagc gaaaaatgga
     2761 acatgatggt tcactttttc aagcagtagg aattggaaca ttattacagc agccagacga
     2821 tcatgcagct actacatcac tttcttggaa acgtgtaaaa ggatgcaaat ctagtgaaca
     2881 gaatggaatg gagcaaaaga caattatttt aataccctct gatttagcat gtagactgct
     2941 ggggcaatca atggatgaaa gtggattacc acagctgacc agttatgatt gtgaagttaa
     3001 tgctcctata caaggcagca gaaacctact gcggggtgaa gaattactca gagctttgga
     3061 tcaagttaac gacccagctt tcttgtacaa agtggttgat ctcaagcttc gaattctgca
     3121 gtcgacggta ccgcgggccc gggatccacc ggtcgccacc atggtgagca agggcgagga
     3181 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     3241 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     3301 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     3361 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     3421 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     3481 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     3541 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     3601 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     3661 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     3721 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     3781 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     3841 cgccgggatc actctcggca tggacgagct gtacaagtaa agcggccgcg actctagatc
     3901 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
     3961 cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct
     4021 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     4081 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaaggcgt aaattgtaag
     4141 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     4201 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     4261 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     4321 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     4381 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     4441 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     4501 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     4561 gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac
     4621 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     4681 ctgataaatg cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg
     4741 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     4801 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     4861 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     4921 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     4981 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     5041 tgaggaggct tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg
     5101 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     5161 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     5221 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     5281 gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     5341 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     5401 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     5461 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     5521 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     5581 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag
     5641 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     5701 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     5761 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     5821 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     5881 tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg
     5941 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     6001 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     6061 gagttcttcg cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa
     6121 ggaacccgcg ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt
     6181 gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc
     6241 cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt
     6301 gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac
     6361 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     6421 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     6481 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     6541 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     6601 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     6661 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     6721 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     6781 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     6841 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     6901 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     6961 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     7021 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     7081 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     7141 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     7201 attaccgcca tgcattagtt attaatagta atcaattacg gggtcattag ttcatagccc
     7261 atatatggag ttccg
//
