LOCUS       sequence-252381-        8501 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral plasmid for expression of hypoxia-responsive enhanced 
            green fluorescent protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8501)
  AUTHORS   Janska L, Anandi L, Kirchberger NC, Marinkovic ZS, Schachtner LT, 
            Guzelsoy G, Carmona-Fontaine C
  TITLE     The MEMIC is an ex vivo system to model the complexity of the tumor 
            microenvironment.
  JOURNAL   Dis Model Mech. 2021 Aug 1;14(8). pii: 271783. doi: 
            10.1242/dmm.048942. Epub 2021 Aug 18.
  PUBMED    34407185
REFERENCE   2  (bases 1 to 8501)
  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: "Dis Model 
            Mech."; date: "2021-08-1"; volume: "14(8). pii: 271783. doi"; pages:
            " 10.1242/dmm.048942. Epub 2021 Aug 18"
COMMENT     Sequence Label: pLenti-5HRE-GFP PuroR
FEATURES             Location/Qualifiers
     source          1..8501
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..181
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    228..353
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    850..1083
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1268..1312
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    1610..1727
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2051..2089
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2086..2110
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2145..2154
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2151..2153,2154..2156,2157..2867,2868..2873,
                     2874..2996)
                     /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(2196..2217)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2457..2476)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2804..2825
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3006..3035
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(3071..3088)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3077..3301
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3368..3867
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3403..3426)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3800..3819
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3888..4487
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3888..3907)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4384..4404
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    4556..5144
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4609..4629)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5027..5038)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5147..5163)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5148..5164)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5673..5853
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(5915..6503)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5995..6014)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6674..7465,7466..7534))
                     /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     7297..7316
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7535..7639)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7714..7733)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7905..8284
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        8286..8484
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
ORIGIN
        1 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
       61 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
      121 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
      181 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca
      241 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc
      301 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta
      361 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa
      421 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc
      481 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc
      541 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg
      601 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc
      661 aaaacaaaag taagaccacc gcacagcaag cggccggccg ctgatcttca gacctggagg
      721 aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga
      781 accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc
      841 agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc
      901 agcgtcaatg acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca
      961 gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg
     1021 catcaagcag ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct
     1081 cctggggatt tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc
     1141 tagttggagt aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga
     1201 cagagaaatt aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca
     1261 gcaagaaaag aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg
     1321 gtttaacata acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt
     1381 ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata
     1441 ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg
     1501 aatagaagaa gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc
     1561 ggcactgcgt gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa
     1621 aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca
     1681 tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca
     1741 gggacagcag agatccagtt tggttagtac cgggcccggg tcgactagtg atcagtgtga
     1801 gggagtgtaa agctggtttt cggtaccgag cttttctcga gccacagtgc atacgtgggc
     1861 tccaacaggt cctcttgtcg agccacagtg catacgtggg ctccaacagg tcctcttgtc
     1921 gagccacagt gcatacgtgg gctccaacag gtcctcttgt cgagccacag tgcatacgtg
     1981 ggctccaaca ggtcctcttg tcgagccaca gtgcatacgt gggctccaac aggtcctctt
     2041 gtcgagatct ggtaggcgtg tacggtggga ggtctatata agcagagctc gtttagtgaa
     2101 ccgtcagatc actagaagct tggcattccg gtactgttgg taaagccacc atggtgagca
     2161 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     2221 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     2281 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     2341 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     2401 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     2461 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     2521 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     2581 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     2641 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     2701 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     2761 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     2821 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagaag ctgagccacg
     2881 gcttcccgcc ggaggtggag gagcaggatg atggcacgct gcccatgtct tgtgcccagg
     2941 agagcgggat ggaccgtcac cctgcagcct gtgcttctgc taggatcaat gtgtaggcgg
     3001 ccgcagaaca aaaactcatc tcagaagagg atctgaatgg ggccgcatag tctagaagct
     3061 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     3121 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     3181 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     3241 agcaaggggg aggattggga agacaatagc aggcatgctg gggatggccc gggctctatg
     3301 gcttctgagg cggaaagaac cagctgggga cccgacatta gcgctacagc ttaagcggaa
     3361 ttctaccggg taggggaggc gcttttccca aggcagtctg gagcatgcgc tttagcagcc
     3421 ccgctgggca cttggcgcta cacaagtggc ctctggcctc gcacacattc cacatccacc
     3481 ggtaggcgcc aaccggctcc gttctttggt ggccccttcg cgccaccttc tactcctccc
     3541 ctagtcagga agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa
     3601 gtagcacgtc tcactagtct cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta
     3661 ggcctttggg gcagcggcca atagcagctt tgctccttcg ctttctgggc tcagaggctg
     3721 ggaaggggtg ggtccggggg cgggctcagg ggcgggctca ggggcggggc gggcgcccga
     3781 aggtcctccg gaggcccggc attctgcacg cttcaaaagc gcacgtctgc cgcgctgttc
     3841 tcctcttcct catctccggg cctttcgacc tgcagcccaa gcttaccatg accgagtaca
     3901 agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc accctcgccg
     3961 ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc
     4021 gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt
     4081 gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg
     4141 gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg
     4201 cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc
     4261 tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc
     4321 tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc
     4381 cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc
     4441 ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgaccg cgtctggaac
     4501 aataaacgtt gttgtttggg gttgaattac tcttctagac cgcggcgcgc ccgataatca
     4561 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt
     4621 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc
     4681 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc
     4741 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg
     4801 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc
     4861 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg
     4921 cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg
     4981 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc
     5041 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct
     5101 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accgtcgacc
     5161 tcgatcgaga cctagaaaaa catggagcaa tcacaagtag caatacagca gctaccaatg
     5221 ctgattgtgc ctggctagaa gcacaagagg aggaggaggt gggttttcca gtcacacctc
     5281 aggtaccttt aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag
     5341 aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatatc cttgatctgt
     5401 ggatctacca cacacaaggc tacttccctg attggcagaa ctacacacca gggccaggga
     5461 tcagatatcc actgaccttt ggatggtgct acaagctagt accagttgag caagagaagg
     5521 tagaagaagc caatgaagga gagaacaccc gcttgttaca ccctgtgagc ctgcatggga
     5581 tggatgaccc ggagagagaa gtattagagt ggaggtttga cagccgccta gcatttcatc
     5641 acatggcccg agagctgcat ccggactgta ctgggtctct ctggttagac cagatctgag
     5701 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
     5761 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
     5821 gaccctttta gtcagtgtgg aaaatctcta gcagcatgtg agcaaaaggc cagcaaaagg
     5881 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5941 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     6001 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     6061 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     6121 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     6181 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     6241 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     6301 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     6361 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6421 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6481 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     6541 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     6601 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6661 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6721 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     6781 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     6841 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6901 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     6961 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     7021 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     7081 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     7141 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     7201 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     7261 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     7321 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     7381 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     7441 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     7501 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     7561 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     7621 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
     7681 gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     7741 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
     7801 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
     7861 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac
     7921 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
     7981 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
     8041 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
     8101 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
     8161 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
     8221 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
     8281 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
     8341 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
     8401 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
     8461 acggtgggag gtctatataa gcagcgcgtt ttgcctgtac t
//
