LOCUS       sequence-273416-        7520 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV-phlFo-phlFR-2A-mCherry mammalian synthetic gene circuit.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7520)
  AUTHORS   Szenk M, Yim T, Balazsi G
  TITLE     Multiplexed Gene Expression Tuning with Orthogonal Synthetic Gene 
            Circuits.
  JOURNAL   ACS Synth Biol. 2020 Apr 17;9(4):930-939. doi: 
            10.1021/acssynbio.9b00534. Epub 2020 Mar 23.
  PUBMED    32167761
REFERENCE   2  (bases 1 to 7520)
  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.9b00534"; journalName: "ACS Synth Biol"; date: 
            "2020-04-17- 17"; volume: "9"; issue: "4"; pages: "930-939"
COMMENT     Sequence Label: phlFs-mLin
FEATURES             Location/Qualifiers
     source          1..7520
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          1033..1605
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     CDS             2234..2254
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2261..2314
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             2315..3025
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2460..2478)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2728..2747
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3099..3119)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     primer_bind     complement(3472..3489)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3478..3702
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3835..3854)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3986..4033
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             4041..5063
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4041..5063
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    5193..5314
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5230..5249)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5284..5303
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5363..5379)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5363..5379)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5376..5398)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5387..5403
                     /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(5411..5417,5418..5435,5436..5441))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5456..5477
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5594..5611)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5765..6353)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5845..5864)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6524..7315,7316..7384))
                     /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     7147..7166
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7385..7489)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcaatgatac gaaacgtacc gtatcgttaa
      841 ggttcagatc gctgttttga cctccataga agacaccggg accgatccag cctccggact
      901 ctagcgttta aacttaagct tggtacccgg ggatcctcta gggcctctga gctattccag
      961 aagtagtgaa gaggcttttt tggaggccta ggcttttgca aaaagctccg gatcgatcct
     1021 gagaacttca gggtgagttt ggggaccctt gattgttctt tctttttcgc tattgtaaaa
     1081 ttcatgttat atggaggggg caaagttttc agggtgttgt ttagaatggg aagatgtccc
     1141 ttgtatcacc atggaccctc atgataattt tgtttctttc actttctact ctgttgacaa
     1201 ccattgtctc ctcttatttt cttttcattt tctgtaactt tttcgttaaa ctttagcttg
     1261 catttgtaac gaatttttaa attcactttt gtttatttgt cagattgtaa gtactttctc
     1321 taatcacttt tttttcaagg caatcagggt atattatatt gtacttcagc acagttttag
     1381 agaacaattg ttataattaa atgataaggt agaatatttc tgcatataaa ttctggctgg
     1441 cgtggaaata ttcttattgg tagaaacaac tacatcctgg tcatcatcct gcctttctct
     1501 ttatggttac aatgatatac actgtttgag atgaggataa aatactctga gtccaaaccg
     1561 ggcccctctg ctaaccatgt tcatgccttc ttctttttcc tacaggtcct gcaggaatag
     1621 gaccatggcc cggaccccct ctagaagctc tatcggcagc ctgagaagcc cccacaccca
     1681 caaggccatc ctgaccagca ccatcgagat cctgaaagag tgcggctaca gcggcctgag
     1741 catcgagtct gtggctagaa gggctggcgc ctccaagccc accatctacc ggtggtggac
     1801 aaacaaggcc gccctgatcg ccgaggtgta cgagaacgag agcgagcagg tccgaaagtt
     1861 ccccgacctg ggcagcttca aggccgatct ggacttcctg ctgcggaacc tgtggaaagt
     1921 gtggcgggaa accatctgcg gcgaggcctt cagatgcgtg atcgctgagg cccagctgga
     1981 ccctgccacc ctgacacagc tgaaggacca gttcatggaa cggcggagag agatgcccaa
     2041 gaaactggtg gaaaacgcca tcagcaacgg cgagctgccc aaggacacca accgggaact
     2101 gctgctggat atgatcttcg gcttctgctg gtacagactg ctgaccgagc agctgaccgt
     2161 ggaacaggat atcgaggaat tcacctttct gctgatcaac ggcgtgtgcc ccggcaccca
     2221 gagagccagc ccccccaaga aaaagcggaa agtggctagc gagggcagag gaagtcttct
     2281 aacatgcggt gacgtggagg agaatcccgg ccctatggtg agcaagggcg aggaggataa
     2341 catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg
     2401 ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc
     2461 caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca
     2521 gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa
     2581 gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt
     2641 ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct
     2701 gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga
     2761 ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag
     2821 gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa
     2881 gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca
     2941 caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg
     3001 cggcatggac gagctgtaca agtaaacttg cattcacgcg gccgcaatca acctctggat
     3061 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     3121 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     3181 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     3241 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     3301 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     3361 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     3421 tccgtggtgt tgtcctcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg
     3481 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     3541 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     3601 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     3661 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     3721 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     3781 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     3841 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     3901 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3961 attagggtga tggttcacgt acctagaagt tcctattccg aagttcctat tctctagaaa
     4021 gtataggaac ttccttggcc aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc
     4081 tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc
     4141 gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg
     4201 atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc
     4261 cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc tcccgccgtg
     4321 cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg
     4381 tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc
     4441 cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg
     4501 ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg
     4561 cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg
     4621 tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca
     4681 ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct
     4741 ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg
     4801 agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt gaccaactct
     4861 atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg
     4921 caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg
     4981 ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca
     5041 ctcgtccgag ggcaaaggaa tagcacgtac tacgagattt cgattccacc gccgccttct
     5101 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     5161 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     5221 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     5281 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     5341 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     5401 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     5461 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     5521 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     5581 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     5641 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     5701 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     5761 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     5821 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     5881 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     5941 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     6001 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     6061 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     6121 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     6181 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     6241 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     6301 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     6361 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     6421 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     6481 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     6541 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     6601 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     6661 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     6721 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     6781 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     6841 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     6901 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     6961 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     7021 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     7081 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     7141 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     7201 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     7261 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     7321 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     7381 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     7441 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     7501 gaaaagtgcc acctgacgtc
//
