LOCUS       sequence-296026-        7528 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV-phlFo2-phlFR-2A-mCherry mammalian synthetic gene circuit.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7528)
  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 7528)
  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: phlFd-mLin
FEATURES             Location/Qualifiers
     source          1..7528
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(55..74)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        246..625
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        626..829
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     780..800
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          1052..1624
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     CDS             2253..2273
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2280..2333
                     /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             2334..3044
                     /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(2479..2497)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2747..2766
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3118..3138)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     primer_bind     complement(3491..3508)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3497..3721
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3854..3873)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4005..4052
                     /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             4060..5082
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4060..5082
                     /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    5212..5333
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5249..5268)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5303..5322
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5382..5398)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5382..5398)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5395..5417)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5406..5422
                     /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(5430..5436,5437..5454,5455..5460))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5475..5496
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5613..5630)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5784..6372)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5864..5883)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6543..7334,7335..7403))
                     /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     7166..7185
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7404..7508)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat
       61 ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc
      121 tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat
      181 gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac
      241 gcgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca
      301 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
      361 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
      421 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
      481 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
      541 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
      601 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg
      661 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
      721 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac
      781 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcaatgata cgaaacgtac
      841 cgtatcgtta aggttcagat catgatacga aacgtaccgt atcgttaagg tgctccggga
      901 ccgatccagc ctccggactc tagcgtttaa acttaagctt ggtacccggg gatcctctag
      961 ggcctctgag ctattccaga agtagtgaag aggctttttt ggaggcctag gcttttgcaa
     1021 aaagctccgg atcgatcctg agaacttcag ggtgagtttg gggacccttg attgttcttt
     1081 ctttttcgct attgtaaaat tcatgttata tggagggggc aaagttttca gggtgttgtt
     1141 tagaatggga agatgtccct tgtatcacca tggaccctca tgataatttt gtttctttca
     1201 ctttctactc tgttgacaac cattgtctcc tcttattttc ttttcatttt ctgtaacttt
     1261 ttcgttaaac tttagcttgc atttgtaacg aatttttaaa ttcacttttg tttatttgtc
     1321 agattgtaag tactttctct aatcactttt ttttcaaggc aatcagggta tattatattg
     1381 tacttcagca cagttttaga gaacaattgt tataattaaa tgataaggta gaatatttct
     1441 gcatataaat tctggctggc gtggaaatat tcttattggt agaaacaact acatcctggt
     1501 catcatcctg cctttctctt tatggttaca atgatataca ctgtttgaga tgaggataaa
     1561 atactctgag tccaaaccgg gcccctctgc taaccatgtt catgccttct tctttttcct
     1621 acaggtcctg caggaatagg accatggccc ggaccccctc tagaagctct atcggcagcc
     1681 tgagaagccc ccacacccac aaggccatcc tgaccagcac catcgagatc ctgaaagagt
     1741 gcggctacag cggcctgagc atcgagtctg tggctagaag ggctggcgcc tccaagccca
     1801 ccatctaccg gtggtggaca aacaaggccg ccctgatcgc cgaggtgtac gagaacgaga
     1861 gcgagcaggt ccgaaagttc cccgacctgg gcagcttcaa ggccgatctg gacttcctgc
     1921 tgcggaacct gtggaaagtg tggcgggaaa ccatctgcgg cgaggccttc agatgcgtga
     1981 tcgctgaggc ccagctggac cctgccaccc tgacacagct gaaggaccag ttcatggaac
     2041 ggcggagaga gatgcccaag aaactggtgg aaaacgccat cagcaacggc gagctgccca
     2101 aggacaccaa ccgggaactg ctgctggata tgatcttcgg cttctgctgg tacagactgc
     2161 tgaccgagca gctgaccgtg gaacaggata tcgaggaatt cacctttctg ctgatcaacg
     2221 gcgtgtgccc cggcacccag agagccagcc cccccaagaa aaagcggaaa gtggctagcg
     2281 agggcagagg aagtcttcta acatgcggtg acgtggagga gaatcccggc cctatggtga
     2341 gcaagggcga ggaggataac atggccatca tcaaggagtt catgcgcttc aaggtgcaca
     2401 tggagggctc cgtgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct
     2461 acgagggcac ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttcgcct
     2521 gggacatcct gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag caccccgccg
     2581 acatccccga ctacttgaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga
     2641 acttcgagga cggcggcgtg gtgaccgtga cccaggactc ctccctgcag gacggcgagt
     2701 tcatctacaa ggtgaagctg cgcggcacca acttcccctc cgacggcccc gtaatgcaga
     2761 agaagaccat gggctgggag gcctcctccg agcggatgta ccccgaggac ggcgccctga
     2821 agggcgagat caagcagagg ctgaagctga aggacggcgg ccactacgac gctgaggtca
     2881 agaccaccta caaggccaag aagcccgtgc agctgcccgg cgcctacaac gtcaacatca
     2941 agttggacat cacctcccac aacgaggact acaccatcgt ggaacagtac gaacgcgccg
     3001 agggccgcca ctccaccggc ggcatggacg agctgtacaa gtaaacttgc attcacgcgg
     3061 ccgcaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     3121 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     3181 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     3241 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     3301 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     3361 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     3421 gctgttgggc actgacaatt ccgtggtgtt gtcctcgagt ctagagggcc cgtttaaacc
     3481 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     3541 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     3601 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     3661 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     3721 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     3781 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     3841 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     3901 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     3961 ctcgacccca aaaaacttga ttagggtgat ggttcacgta cctagaagtt cctattccga
     4021 agttcctatt ctctagaaag tataggaact tccttggcca aaaagcctga actcaccgcg
     4081 acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc
     4141 tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg
     4201 cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca
     4261 tcggccgcgc tcccgattcc ggaagtgctt gacattgggg aattcagcga gagcctgacc
     4321 tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg
     4381 cccgctgttc tgcagccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc
     4441 cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt
     4501 gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac
     4561 accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc
     4621 cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat
     4681 ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag
     4741 gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac
     4801 ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc
     4861 attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg
     4921 gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa
     4981 atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt
     5041 ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat agcacgtact acgagatttc
     5101 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     5161 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
     5221 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     5281 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     5341 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     5401 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     5461 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     5521 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     5581 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     5641 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     5701 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     5761 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     5821 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     5881 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     5941 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     6001 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     6061 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     6121 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     6181 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     6241 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     6301 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     6361 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     6421 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     6481 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     6541 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     6601 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     6661 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     6721 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     6781 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6841 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     6901 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     6961 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     7021 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     7081 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     7141 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     7201 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     7261 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     7321 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     7381 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     7441 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     7501 gttccgcgca catttccccg aaaagtgc
//
