LOCUS       sequence-296025-        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(53..72)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        244..623
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        624..827
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     778..798
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          1042..1614
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     CDS             2243..2263
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2270..2323
                     /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             2324..3034
                     /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(2469..2487)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2737..2756
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3108..3128)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     primer_bind     complement(3481..3498)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3487..3711
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3844..3863)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3995..4042
                     /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             4050..5072
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4050..5072
                     /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    5202..5323
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5239..5258)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5293..5312
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5372..5388)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5372..5388)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5385..5407)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5396..5412
                     /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(5420..5426,5427..5444,5445..5450))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5465..5486
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5603..5620)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5774..6362)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5854..5873)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6533..7324,7325..7393))
                     /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     7156..7175
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7394..7498)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct
       61 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg
      121 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga
      181 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc
      241 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
      301 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      361 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      421 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      481 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      541 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      601 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
      661 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
      721 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
      781 aaatgggcgg taggcgtgta cggtgggagg tctatataag caatgatacg aaacgtaccg
      841 tatcgttaag gttcagatcg ctgttttgac ctccatagaa gacaccggga ccgatccagc
      901 ctccggactc tagcgtttaa acttaagctt ggtacccggg gatcctctag ggcctctgag
      961 ctattccaga agtagtgaag aggctttttt ggaggcctag gcttttgcaa aaagctccgg
     1021 atcgatcctg agaacttcag ggtgagtttg gggacccttg attgttcttt ctttttcgct
     1081 attgtaaaat tcatgttata tggagggggc aaagttttca gggtgttgtt tagaatggga
     1141 agatgtccct tgtatcacca tggaccctca tgataatttt gtttctttca ctttctactc
     1201 tgttgacaac cattgtctcc tcttattttc ttttcatttt ctgtaacttt ttcgttaaac
     1261 tttagcttgc atttgtaacg aatttttaaa ttcacttttg tttatttgtc agattgtaag
     1321 tactttctct aatcactttt ttttcaaggc aatcagggta tattatattg tacttcagca
     1381 cagttttaga gaacaattgt tataattaaa tgataaggta gaatatttct gcatataaat
     1441 tctggctggc gtggaaatat tcttattggt agaaacaact acatcctggt catcatcctg
     1501 cctttctctt tatggttaca atgatataca ctgtttgaga tgaggataaa atactctgag
     1561 tccaaaccgg gcccctctgc taaccatgtt catgccttct tctttttcct acaggtcctg
     1621 caggaatagg accatggccc ggaccccctc tagaagctct atcggcagcc tgagaagccc
     1681 ccacacccac aaggccatcc tgaccagcac catcgagatc ctgaaagagt gcggctacag
     1741 cggcctgagc atcgagtctg tggctagaag ggctggcgcc tccaagccca ccatctaccg
     1801 gtggtggaca aacaaggccg ccctgatcgc cgaggtgtac gagaacgaga gcgagcaggt
     1861 ccgaaagttc cccgacctgg gcagcttcaa ggccgatctg gacttcctgc tgcggaacct
     1921 gtggaaagtg tggcgggaaa ccatctgcgg cgaggccttc agatgcgtga tcgctgaggc
     1981 ccagctggac cctgccaccc tgacacagct gaaggaccag ttcatggaac ggcggagaga
     2041 gatgcccaag aaactggtgg aaaacgccat cagcaacggc gagctgccca aggacaccaa
     2101 ccgggaactg ctgctggata tgatcttcgg cttctgctgg tacagactgc tgaccgagca
     2161 gctgaccgtg gaacaggata tcgaggaatt cacctttctg ctgatcaacg gcgtgtgccc
     2221 cggcacccag agagccagcc cccccaagaa aaagcggaaa gtggctagcg agggcagagg
     2281 aagtcttcta acatgcggtg acgtggagga gaatcccggc cctatggtga gcaagggcga
     2341 ggaggataac atggccatca tcaaggagtt catgcgcttc aaggtgcaca tggagggctc
     2401 cgtgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac
     2461 ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttcgcct gggacatcct
     2521 gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag caccccgccg acatccccga
     2581 ctacttgaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga
     2641 cggcggcgtg gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa
     2701 ggtgaagctg cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagaccat
     2761 gggctgggag gcctcctccg agcggatgta ccccgaggac ggcgccctga agggcgagat
     2821 caagcagagg ctgaagctga aggacggcgg ccactacgac gctgaggtca agaccaccta
     2881 caaggccaag aagcccgtgc agctgcccgg cgcctacaac gtcaacatca agttggacat
     2941 cacctcccac aacgaggact acaccatcgt ggaacagtac gaacgcgccg agggccgcca
     3001 ctccaccggc ggcatggacg agctgtacaa gtaaacttgc attcacgcgg ccgcaatcaa
     3061 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     3121 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     3181 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc
     3241 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg
     3301 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc
     3361 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
     3421 actgacaatt ccgtggtgtt gtcctcgagt ctagagggcc cgtttaaacc cgctgatcag
     3481 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     3541 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     3601 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     3661 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     3721 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa
     3781 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3841 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3901 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3961 aaaaacttga ttagggtgat ggttcacgta cctagaagtt cctattccga agttcctatt
     4021 ctctagaaag tataggaact tccttggcca aaaagcctga actcaccgcg acgtctgtcg
     4081 agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg
     4141 aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata
     4201 gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc
     4261 tcccgattcc ggaagtgctt gacattgggg aattcagcga gagcctgacc tattgcatct
     4321 cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc
     4381 tgcagccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg
     4441 ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat
     4501 gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg
     4561 cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc
     4621 ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa
     4681 cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca
     4741 tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga
     4801 ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg
     4861 accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc
     4921 gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca
     4981 gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac
     5041 gccccagcac tcgtccgagg gcaaaggaat agcacgtact acgagatttc gattccaccg
     5101 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     5161 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     5221 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     5281 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     5341 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     5401 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     5461 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     5521 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     5581 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     5641 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     5701 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     5761 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     5821 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     5881 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     5941 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     6001 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     6061 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     6121 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     6181 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     6241 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     6301 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     6361 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     6421 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     6481 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     6541 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     6601 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     6661 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     6721 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     6781 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     6841 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     6901 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     6961 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     7021 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     7081 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     7141 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     7201 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     7261 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     7321 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     7381 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     7441 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     7501 catttccccg aaaagtgcca
//
