LOCUS       sequence-225154-        7175 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sfGFP expressed from hypoxia-inducible miniTK promoter and dsRed 
            expressed from consitutive CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7175)
  AUTHORS   Ede C, Chen X, Lin MY, Chen YY
  TITLE     Quantitative Analyses of Core Promoters Enable Precise Engineering 
            of Regulated Gene Expression in Mammalian Cells.
  JOURNAL   ACS Synth Biol. 2016 May 20;5(5):395-404. doi: 
            10.1021/acssynbio.5b00266. Epub 2016 Mar 1.
  PUBMED    26883397
REFERENCE   2  (bases 1 to 7175)
  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: 
            "10.1021/acssynbio.5b00266"; journalName: "ACS Synth Biol"; date: 
            "2016-05-20- 20"; volume: "5"; issue: "5"; pages: "395-404"
COMMENT     Sequence Label: 4xHRE_miniTK-sfGFP-CMV_dsRed Exp
FEATURES             Location/Qualifiers
     source          1..7175
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     249..268
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        249..267
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      288..297
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             294..971
                     /codon_start=1
                     /product="rapidly maturing tetrameric variant of DsRed 
                     fluorescent protein (Bevis and Glick, 2002)"
                     /label=DsRed-Express
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRHH
                     LFL"
     primer_bind     complement(424..442)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     692..711
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     886..909
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    1003..1227
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1273..1701
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1360..1379)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1570..1591
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1710..1730)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1715..2044
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1895..2030
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1957..1976
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2111..2905
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2165..2184)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2775..2794
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3079..3200
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3116..3135)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3170..3189
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3249..3265)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3249..3265)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3262..3284)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3273..3289
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3297..3303,3304..3321,3322..3327))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3342..3363
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3480..3497)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3651..4239)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3731..3750)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4410..5201,5202..5270))
                     /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     5033..5052
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5271..5375)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5618..5680
                     /label=Mini-TK promoter
                     /note="minimal herpes simplex virus (HSV) thymidine kinase 
                     promoter"
     polyA_signal    6469..6693
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        6796..7175
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc ttactggctt
      241 atcgaaatta atacgactca ctatagggag acccaagctg gctagccgcc accatggcct
      301 cctccgagga cgtcatcaag gagttcatgc gcttcaaggt gcgcatggag ggctccgtga
      361 acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga
      421 ccgccaagct gaaggtgacc aagggcggcc ccctgccctt cgcctgggac atcctgtccc
      481 cccagttcca gtacggctcc aaggtgtacg tgaagcaccc cgccgacatc cccgactaca
      541 agaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg
      601 gcgtggtgac cgtgacccag gactcctccc tgcaggacgg ctccttcatc tacaaggtga
      661 agttcatcgg cgtgaacttc ccctccgacg gccccgtaat gcagaagaag actatgggct
      721 gggaggcctc caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc gagatccaca
      781 aggccctgaa gctgaaggac ggcggccact acctggtgga gttcaagtcc atctacatgg
      841 ccaagaagcc cgtgcagctg cccggctact actacgtgga ctccaagctg gacatcacct
      901 cccacaacga ggactacacc atcgtggagc agtacgagcg cgccgagggc cgccaccacc
      961 tgttcctgta gacggctgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     1021 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     1081 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     1141 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     1201 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta
     1261 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     1321 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     1381 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     1441 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     1501 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     1561 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     1621 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     1681 aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc
     1741 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     1801 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     1861 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     1921 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     1981 ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     2041 caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg
     2101 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     2161 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     2221 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     2281 gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     2341 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     2401 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     2461 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     2521 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     2581 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg
     2641 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     2701 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     2761 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     2821 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     2881 tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg
     2941 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     3001 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     3061 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     3121 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     3181 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     3241 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     3301 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     3361 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     3421 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     3481 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     3541 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     3601 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     3661 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     3721 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     3781 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     3841 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     3901 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     3961 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4021 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4081 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4141 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4201 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     4261 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     4321 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     4381 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     4441 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     4501 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     4561 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     4621 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     4681 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     4741 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     4801 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     4861 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     4921 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     4981 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     5041 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     5101 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     5161 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     5221 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     5281 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     5341 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     5401 gacgtcgacg gatcgggaga tctttgtatt taattaagac cttgagtacg tgcgtctctg
     5461 cacgtatggc gattaagacc ttgagtacgt gcgtctctgc acgtatggcg attaagacct
     5521 tgagtacgtg cgtctctgca cgtatggcga ttaagacctt gagtacgtgc gtctctgcac
     5581 gtatggcgga tcttcgcgat tacgcaataa tggtaccttc gcatattaag gtgacgcgtg
     5641 tggcctcgaa caccgagcga ccctgcagcg acccgcttaa aagcttgaat tctctagcgc
     5701 cgccaccatg agtaaaggtg aagaactgtt caccggtgtt gttccgatcc tggttgaact
     5761 ggatggtgat gttaacggcc acaaattctc tgttcgtggt gaaggtgaag gtgatgcaac
     5821 caacggtaaa ctgaccctga aattcatctg cactaccggt aaactgccgg ttccatggcc
     5881 gactctggtg actaccctga cctatggtgt tcagtgtttt tctcgttacc cggatcacat
     5941 gaagcagcat gatttcttca aatctgcaat gccggaaggt tatgtacagg agcgcaccat
     6001 ttctttcaaa gacgatggca cctacaaaac ccgtgcagag gttaaatttg aaggtgatac
     6061 tctggtgaac cgtattgaac tgaaaggcat tgatttcaaa gaggacggca acatcctggg
     6121 ccacaaactg gaatataact tcaactccca taacgtttac atcaccgcag acaaacagaa
     6181 gaacggtatc aaagctaact tcaaaattcg ccataacgtt gaagacggta gcgtacagct
     6241 ggcggaccac taccagcaga acactccgat cggtgatggt ccggttctgc tgccggataa
     6301 ccactacctg tccacccagt ctaaactgtc caaagacccg aacgaaaagc gcgaccacat
     6361 ggtgctgctg gagttcgtta ctgcagcagg tatcacgcac ggcatggatg aactctacaa
     6421 ataagcggcc gcctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta
     6481 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     6541 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     6601 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     6661 gcaggcatgc tggggatgcg gtgggctcta tggccgcgga attagcctat gttttttgga
     6721 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg
     6781 ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt
     6841 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     6901 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     6961 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     7021 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     7081 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     7141 agtacatcta cgtattagtc atcgctatta ccatg
//
