LOCUS       sequence-391954-        6317 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  LPUtopia-7 matching RMCE donor plasmid with GFP Positive-Feedback 
            circuit (mPF-GFP). Use BlastR for positive selection and HSV-TK for 
            negative selection. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6317)
  AUTHORS   Wan Y, Cohen J, Szenk M, Farquhar KS, Coraci D, Krzyszton R, Azukas 
            J, Van Nest N, Smashnov A, Chern YJ, De Martino D, Nguyen LC, Bien 
            H, Bravo-Cordero JJ, Chan CH, Rosner MR, Balazsi G
  TITLE     Nonmonotone invasion landscape by noise-aware control of metastasis 
            activator levels.
  JOURNAL   Nat Chem Biol. 2023 Jul;19(7):887-899. doi: 
            10.1038/s41589-023-01344-z. Epub 2023 May 25.
  PUBMED    37231268
REFERENCE   2  (bases 1 to 6317)
  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: 
            "10.1038/s41589-023-01344-z"; journalName: "Nat Chem Biol"; date: 
            "2023-07"; volume: "19"; issue: "7"; pages: "887-899"
COMMENT     Sequence Label: pUt-mPF-GFP
FEATURES             Location/Qualifiers
     source          1..6317
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    65..459
                     /label=beta-globin poly(A)
                     /note="human beta-globin polyadenylation signal"
     promoter        complement(900..918)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(910..956)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(1003..1205)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     enhancer        1207..1510
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    1554..1601
                     /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)."
     rep_origin      complement(1613..2201)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1693..1712)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2234..2733
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2269..2292)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2666..2685
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     5'UTR           2774..2813
                     /label=HSV TK 5'-UTR
                     /note="5' untranslated region from the herpes simplex virus
                     thymidine kinase gene"
     CDS             2831..3961
                     /codon_start=1
                     /product="herpes simplex virus thymidine kinase"
                     /label=HSV TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MASYPCHQHASAFDQAARSRGHNNRRTALRPRRQQKATEVRLEQK
                     MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWRVLGASETIANIYTTQ
                     HRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGSSHAPPPALTLIFDR
                     HPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQR
                     PGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWREDWGQLSGAAVPPQGAEPQSNAGPR
                     PHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPAGCRDAL
                     LQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
     primer_bind     complement(3959..3978)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4003..4051
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    4075..4108
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     promoter        4150..4464
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    4158..4176
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4194..4212
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4229..4247
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4265..4283
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4301..4319
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4336..4354
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4372..4390
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             join(4469..5086,5087..5092,5093..5128,5129..5131,5132..5167,
                     5168..5170,5171..5206,5207..5212)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             5222..5278
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(5279..5281,5282..5284,5285..5998)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(5279..5281,5282..5284,5285..5995)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5324..5345)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5585..5604)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(6092..6109)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    join(6098..6317,1..5)
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 tatggcttct gaggcggaaa cgcgctaaag caagtaaaac ctctacaaat gtggtatgga
       61 attctaaaat acagcatagc aaaactttaa cctccaaatc aagcctctac ttgaatcctt
      121 ttctgaggga tgaataaggc ataggcatca ggggctgttg ccaatgtgca ttagctgttt
      181 gcagcctcac cttctttcat ggagtttaag atatagtgta ttttcccaag gtttgaacta
      241 gctcttcatt tctttatgtt ttaaatgcac tgacctccca cattcccttt ttagtaaaat
      301 attcagaaat aatttaaata catcattgca atgaaaataa atgtttttta ttaggcagaa
      361 tccagatgct caaggccctt cataatatcc cccagtttag tagttggact tagggaacaa
      421 aggaaccttt aatagaaatt ggacagcaag aaagcgagct tctagcttta gttcctggtg
      481 tacttgaggg ggatgagttc ctcaatggtg gttttgacca gcttgccatt catctcaatg
      541 agcacaaagc agtcaggagc atagtcagag atgagctctc tgcacatgcc acaggggctg
      601 accaccctga tggatctgtc cacctcatca gagtaggggt gcctgacagc cacaatggtg
      661 tcaaagtcct tctgcccgtt gctcacagca gacccaatgg caatggcttc agcacagaca
      721 gtgaccctgc caatgtaggc ctcaatgtgg acagcagaga tgatctcccc agtcttggtc
      781 ctgatggccg ccccgacatg gtgcttgttg tcctcataga gcatggtgat cttctcagtg
      841 gcgacctcca ccagctccag atcctgctga gagatgttga aggtcttcat ggtggccctc
      901 ctatagtgag tcgtattata ctatgccgat atactatgcc gatgattaat tgtcaaaaca
      961 gcgtggatgg cgtctccagc ttatctgacg gttcactaaa cgagctctgc ttatatagac
     1021 ctcccaccgt acacgcctac cgcccatttg cgtcaatggg gcggagttgt tacgacattt
     1081 tggaaagtcc cgttgattta ctagtcaaaa caaactccca ttgacgtcaa tggggtggag
     1141 acttggaaat ccccgtgagt caaaccgcta tccacgccca ttgatgtact gccaaaaccg
     1201 catcatcatg gtaatagcga tgactaatac gtagatgtac tgccaagtag gaaagtccca
     1261 taaggtcatg tactgggcat aatgccaggc gggccattta ccgtcattga cgtcaatagg
     1321 gggcgtactt ggcatatgat acacttgatg tactgccaag tgggcagttt accgtaaata
     1381 ctccacccat tgacgtcaat ggaaagtccc tattggcgtt actatgggaa catacgtcat
     1441 tattgacgtc aatgggcggg ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag
     1501 ttatgtaacg cctgcaggtt aattaagaac atgtgagcaa aaggccagca aaagaagttc
     1561 ctattccgaa gttcctattc tctagaaagt ataggaactt cttgctggcg tttttccata
     1621 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     1681 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     1741 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     1801 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     1861 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     1921 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     1981 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     2041 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     2101 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     2161 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     2221 ctacggttct accgggtagg ggaggcgctt ttcccaaggc agtctggagc atgcgcttta
     2281 gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac acattccaca
     2341 tccaccggta ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc accttctact
     2401 cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa
     2461 atggaagtag cacgtctcac tagtctcgtg cagatggaca gcaccgctga gcaatggaag
     2521 cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt ctgggctcag
     2581 aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg cggggcgggc
     2641 gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg
     2701 ctgttctcct cttcctcatc tccgggcctt tcgacctgca gcgacccgct aacagcgtca
     2761 acagcgtgcc gcagatcttg gtggcgtgaa actcccgcac ctcttcggcc agcgccttgt
     2821 agaagcgcgt atggcttcgt acccctgcca tcaacacgcg tctgcgttcg accaggctgc
     2881 gcgttctcgc ggccataaca accgacgtac ggcgttgcgc cctcgccggc aacaaaaagc
     2941 cacggaagtc cgcctggagc agaaaatgcc cacgctactg cgggtttata tagacggtcc
     3001 ccacgggatg gggaaaacca ccaccacgca actgctggtg gccctgggtt cgcgcgacga
     3061 tatcgtctac gtacccgagc cgatgactta ctggcgggtg ttgggggctt ccgagacaat
     3121 cgcgaacatc tacaccacac aacaccgcct cgaccagggt gagatatcgg ccggggacgc
     3181 ggcggtggta atgacaagcg cccagataac aatgggcatg ccttatgccg tgaccgacgc
     3241 cgttctggct cctcatatcg ggggggaggc tgggagctca catgccccgc ccccggccct
     3301 caccctcatc ttcgaccgcc atcccatcgc cgccctcctg tgctacccgg ccgcgcgata
     3361 ccttatgggc agcatgaccc cccaggccgt gctggcgttc gtggccctca tcccgccgac
     3421 cttgcccggc acaaacatcg tgttgggggc ccttccggag gacagacaca tcgaccgcct
     3481 ggccaaacgc cagcgccccg gcgagcggct tgacctggct atgctggccg cgattcgccg
     3541 cgtttatggg ctgcttgcca atacggtgcg gtatctgcag ggcggcgggt cgtggcggga
     3601 ggattgggga cagctttcgg gggcggccgt gccgccccag ggtgccgagc cccagagcaa
     3661 cgcgggccca cgaccccata tcggggacac gttatttacc ctgtttcggg cccccgagtt
     3721 gctggccccc aacggcgacc tgtacaacgt gtttgcctgg gccttggacg tcttggccaa
     3781 acgcctccgt cccatgcacg tctttatcct ggattacgac caatcgcccg ccggctgccg
     3841 ggacgccctg ctgcaactta cctccgggat ggtccagacc cacgtcacca cccccggctc
     3901 cataccgacg atctgcgacc tggcgcgcac gtttgcccgg gagatggggg aggctaactg
     3961 aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga
     4021 ataaaacgca cgggtgttgg gtcgtttgtt cataaacgcg gaagttccta ttccgaagtt
     4081 cctattcttc aaatagtata ggaacttcac gggccagata tacgcgtcga ggccctttcg
     4141 tcttcactcg agtttactcc ctatcagtga tagagaacgt atgtcgagtt tactccctat
     4201 cagtgataga gaacgatgtc gagtttactc cctatcagtg atagagaacg tatgtcgagt
     4261 ttactcccta tcagtgatag agaacgtatg tcgagtttac tccctatcag tgatagagaa
     4321 cgtatgtcga gtttatccct atcagtgata gagaacgtat gtcgagttta ctccctatca
     4381 gtgatagaga acgtatgtcg aggtaggcgt gtacggtggg aggcctatat aagcagagct
     4441 cgtttagtga accgtcagat cgcctggaat gtctagactg gacaagagca aagtcataaa
     4501 cggcgctctg gaattactca atggagtcgg tatcgaaggc ctgacgacaa ggaaactcgc
     4561 tcaaaagctg ggagttgagc agcctaccct gtactggcac gtgaagaaca agcgggccct
     4621 gctcgatgcc ctgccaatcg agatgctgga caggcatcat acccacttct gccccctgga
     4681 aggcgagtca tggcaagact ttctgcggaa caacgccaag tcattccgct gtgctctcct
     4741 ctcacatcgc gacggggcta aagtgcatct cggcacccgc ccaacagaga aacagtacga
     4801 aaccctggaa aatcagctcg cgttcctgtg tcagcaaggc ttctccctgg agaacgcact
     4861 gtacgctctg tccgccgtgg gccactttac actgggctgc gtattggagg aacaggagca
     4921 tcaagtagca aaagaggaaa gagagacacc taccaccgat tctatgcccc cacttctgag
     4981 acaagcaatt gagctgttcg accggcaggg agccgaacct gccttccttt tcggcctgga
     5041 actaatcata tgtggcctgg agaaacagct aaagtgcgaa agcggcgggc cggccgacgc
     5101 ccttgacgat tttgacttag acatgctccc agccgatgcc cttgacgact ttgaccttga
     5161 tatgctgcct gctgacgctc ttgacgattt tgaccttgac atgctccccg ggggaagcgg
     5221 agctactaac ttcagcctgc tgaagcaggc tggagacgtg gaggagaacc ctggacctat
     5281 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     5341 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
     5401 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     5461 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     5521 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     5581 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     5641 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     5701 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     5761 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     5821 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     5881 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     5941 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaagg
     6001 atccactagt ccagtgtggt ggaattctgc agatatccag cacagtggcg gccgctcgag
     6061 tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca
     6121 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     6181 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     6241 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     6301 ggggatgcgg tgggctc
//
