LOCUS       sequence-225156-        7314 bp    DNA     circular SYN 14-APR-2025
DEFINITION  sfGFP expressed from hypoxia-inducible minimal SV40 promoter and 
            dsRed expressed from consitutive CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7314)
  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 7314)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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_minSV40-sfGFP-CMV_dsRed
FEATURES             Location/Qualifiers
     source          1..7314
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      668..677
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             674..1351
                     /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(804..822)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1072..1091
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1266..1289
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    1383..1607
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1653..2081
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1740..1759)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1950..1971
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2090..2110)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2095..2424
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2275..2410
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2491..3285
                     /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(2545..2564)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3155..3174
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3459..3580
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3496..3515)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3550..3569
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3629..3645)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3629..3645)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3642..3664)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3653..3669
                     /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(3677..3683,3684..3701,3702..3707))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3722..3743
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3860..3877)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4031..4619)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4111..4130)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4790..5581,5582..5650))
                     /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     5413..5432
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5651..5755)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5980..6176
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6027..6162
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6988..7212
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagccgcc accatggcct cctccgagga cgtcatcaag gagttcatgc gcttcaaggt
      721 gcgcatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg
      781 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggcggcc ccctgccctt
      841 cgcctgggac atcctgtccc cccagttcca gtacggctcc aaggtgtacg tgaagcaccc
      901 cgccgacatc cccgactaca agaagctgtc cttccccgag ggcttcaagt gggagcgcgt
      961 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg
     1021 ctccttcatc tacaaggtga agttcatcgg cgtgaacttc ccctccgacg gccccgtaat
     1081 gcagaagaag actatgggct gggaggcctc caccgagcgc ctgtaccccc gcgacggcgt
     1141 gctgaagggc gagatccaca aggccctgaa gctgaaggac ggcggccact acctggtgga
     1201 gttcaagtcc atctacatgg ccaagaagcc cgtgcagctg cccggctact actacgtgga
     1261 ctccaagctg gacatcacct cccacaacga ggactacacc atcgtggagc agtacgagcg
     1321 cgccgagggc cgccaccacc tgttcctgta gacggctgtt taaacccgct gatcagcctc
     1381 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     1441 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     1501 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     1561 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     1621 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     1681 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     1741 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     1801 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     1861 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     1921 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     1981 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     2041 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     2101 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     2161 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     2221 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     2281 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     2341 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     2401 tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct
     2461 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     2521 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     2581 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     2641 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     2701 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     2761 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     2821 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     2881 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     2941 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     3001 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     3061 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     3121 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     3181 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     3241 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     3301 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     3361 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     3421 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     3481 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     3541 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     3601 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     3661 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     3721 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     3781 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     3841 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     3901 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     3961 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4021 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4081 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4141 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4201 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4261 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4321 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4381 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4441 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     4501 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4561 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     4621 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4681 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     4741 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     4801 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     4861 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     4921 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     4981 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5041 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     5101 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     5161 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     5221 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5281 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5341 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5401 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     5461 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     5521 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     5581 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     5641 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5701 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5761 ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctttgtatt taattaagac
     5821 cttgagtacg tgcgtctctg cacgtatggc gattaagacc ttgagtacgt gcgtctctgc
     5881 acgtatggcg attaagacct tgagtacgtg cgtctctgca cgtatggcga ttaagacctt
     5941 gagtacgtgc gtctctgcac gtatggcgga tctgcgatct gcatctcaat tagtcagcaa
     6001 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     6061 ctccgcccca tcgctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     6121 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     6181 ttggcattcc ggtactgggt aaagccgaat tcctagcgcc gccaccatga gtaaaggtga
     6241 agaactgttc accggtgttg ttccgatcct ggttgaactg gatggtgatg ttaacggcca
     6301 caaattctct gttcgtggtg aaggtgaagg tgatgcaacc aacggtaaac tgaccctgaa
     6361 attcatctgc actaccggta aactgccggt tccatggccg actctggtga ctaccctgac
     6421 ctatggtgtt cagtgttttt ctcgttaccc ggatcacatg aagcagcatg atttcttcaa
     6481 atctgcaatg ccggaaggtt atgtacagga gcgcaccatt tctttcaaag acgatggcac
     6541 ctacaaaacc cgtgcagagg ttaaatttga aggtgatact ctggtgaacc gtattgaact
     6601 gaaaggcatt gatttcaaag aggacggcaa catcctgggc cacaaactgg aatataactt
     6661 caactcccat aacgtttaca tcaccgcaga caaacagaag aacggtatca aagctaactt
     6721 caaaattcgc cataacgttg aagacggtag cgtacagctg gcggaccact accagcagaa
     6781 cactccgatc ggtgatggtc cggttctgct gccggataac cactacctgt ccacccagtc
     6841 taaactgtcc aaagacccga acgaaaagcg cgaccacatg gtgctgctgg agttcgttac
     6901 tgcagcaggt atcacgcacg gcatggatga actctacaaa taagcggccg cctagagggc
     6961 ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     7021 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     7081 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     7141 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     7201 tgggctctat ggccgcggaa ttagcctatg ttttttggaa caattgcatg aagaatctgc
     7261 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgtt
//
