LOCUS       sequence-177232-        5392 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  hypoxia-responsive enhanced green fluorescent protein (EGFP)-based 
            system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5392)
  AUTHORS   Vordermark D, Shibata T, Brown JM
  TITLE     Green fluorescent protein is a suitable reporter of tumor hypoxia 
            despite an oxygen requirement for chromophore formation.
  JOURNAL   Neoplasia. 2001 Nov-Dec;3(6):527-34.
  PUBMED    11774035
REFERENCE   2  (bases 1 to 5392)
  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"; journalName: "Neoplasia. 
            2001 Nov-Dec"; volume: "3(6)"; pages: "527-34"
COMMENT     Sequence Label: 5HRE/GFP
FEATURES             Location/Qualifiers
     source          1..5392
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        27..65
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     62..86
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      121..130
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(127..129,130..132,133..843,844..849,850..972)
                     /codon_start=1
                     /product="EGFP destabilized by fusion to residues 422-461 
                     of mouse ornithine decarboxylase, giving an in vivo 
                     half-life of ~2 hours"
                     /label=d2EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYKKLSHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV
                     "
     primer_bind     complement(172..193)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(433..452)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     780..801
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             982..1011
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1047..1064)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1053..1277
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1323..1751
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1410..1429)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1620..1641
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1760..1780)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1765..2094
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1945..2080
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2007..2026
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2120..2914
                     /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="MVEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2174..2193)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2784..2803
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3088..3209
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3125..3144)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3179..3198
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3242..3259)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3413..4001)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3493..3512)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4172..4963,4964..5032))
                     /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     4795..4814
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5033..5137)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tccaacaggt cctcttgtcg agatctggta ggcgtgtacg gtgggaggtc tatataagca
       61 gagctcgttt agtgaaccgt cagatcacta gaagcttggc attccggtac tgttggtaaa
      121 gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
      181 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
      241 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
      301 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
      361 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
      421 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
      481 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
      541 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
      601 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
      661 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
      721 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
      781 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
      841 aagaagctga gccacggctt cccgccggag gtggaggagc aggatgatgg cacgctgccc
      901 atgtcttgtg cccaggagag cgggatggac cgtcaccctg cagcctgtgc ttctgctagg
      961 atcaatgtgt aggcggccgc agaacaaaaa ctcatctcag aagaggatct gaatggggcc
     1021 gcatagtcta gaagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     1081 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     1141 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     1201 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     1261 tggcccgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     1321 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     1381 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     1441 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     1501 tagtgcttta cggcacctct cccccaaaaa acttgattag ggtgatggtt cacgtagtgg
     1561 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     1621 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     1681 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     1741 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     1801 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     1861 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     1921 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     1981 ccgcccctag gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     2041 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     2101 cccccccggg aggtccacaa tggttgaaca agatggattg cacgcaggtt ctccggccgc
     2161 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     2221 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     2281 cggtgccctg aatgaactcc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg
     2341 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     2401 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     2461 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     2521 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     2581 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     2641 caaggcgcgt atgcccgacg gcgaggatct cgtcgtgact catggcgatg cctgcttgcc
     2701 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     2761 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     2821 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     2881 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
     2941 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     3001 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     3061 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     3121 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     3181 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgat ctttccgctt
     3241 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     3301 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     3361 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     3421 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     3481 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     3541 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     3601 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     3661 gctgtatgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     3721 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     3781 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     3841 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     3901 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     3961 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     4021 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     4081 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     4141 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     4201 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     4261 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     4321 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     4381 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     4441 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     4501 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     4561 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     4621 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     4681 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     4741 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     4801 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     4861 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgggcaccca
     4921 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     4981 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     5041 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     5101 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     5161 ctgacgtcag atcgacggat cgggagatcg gtaccgagct tttctcgagc cacagtgcat
     5221 acgtgggctc caacaggtcc tcttgtcgag ccacagtgca tacgtgggct ccaacaggtc
     5281 ctcttgtcga gccacagtgc atacgtgggc tccaacaggt cctcttgtcg agccacagtg
     5341 catacgtggg ctccaacagg tcctcttgtc gagccacagt gcatacgtgg gc
//
