LOCUS       sequence-396540-        6283 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 6283)
  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 6283)
  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..6283
                     /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"
     primer_bind     complement(2269..2292)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2631..2650
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     5'UTR           2740..2779
                     /label=HSV TK 5'-UTR
                     /note="5' untranslated region from the herpes simplex virus
                     thymidine kinase gene"
     CDS             2797..3927
                     /codon_start=1
                     /product="herpes simplex virus thymidine kinase"
                     /label=HSV TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MASYPCHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRLEQK
                     MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWQVLGASETIANIYTTQ
                     HRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGSSHAPPPALTLIFDR
                     HPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQR
                     PGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWREDWGQLSGTAVPPQGAEPQSNAGPR
                     PHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPAGCRDAL
                     LQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
     primer_bind     complement(3925..3944)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3969..4017
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    4041..4074
                     /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        4116..4430
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    4124..4142
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4160..4178
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4195..4213
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4231..4249
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4267..4285
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4302..4320
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4338..4356
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             join(4435..5052,5053..5058,5059..5094,5095..5097,5098..5133,
                     5134..5136,5137..5172,5173..5178)
                     /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             5188..5244
                     /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(5245..5247,5248..5250,5251..5964)
                     /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(5245..5247,5248..5250,5251..5961)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5290..5311)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5551..5570)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(6058..6075)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    join(6064..6283,1..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 gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc ggaggcccgg cattctgcac
     2641 gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc tcatctccgg gcctttcgac
     2701 ctgcagcgac ccgcttaaca gcgtcaacag cgtgccgcag atcttggtgg cgtgaaactc
     2761 ccgcacctct tcggcaagcg ccttgtagaa gcgcgtatgg cttcgtaccc ctgccatcaa
     2821 cacgcgtctg cgttcgacca ggctgcgcgt tctcgcggcc atagcaaccg acgtacggcg
     2881 ttgcgccctc gccggcagca agaagccacg gaagtccgcc tggagcagaa aatgcccacg
     2941 ctactgcggg tttatataga cggtcctcac gggatgggga aaaccaccac cacgcaactg
     3001 ctggtggccc tgggttcgcg cgacgatatc gtctacgtac ccgagccgat gacttactgg
     3061 caggtgctgg gggcttccga gacaatcgcg aacatctaca ccacacaaca ccgcctcgac
     3121 cagggtgaga tatcggccgg ggacgcggcg gtggtaatga caagcgccca gataacaatg
     3181 ggcatgcctt atgccgtgac cgacgccgtt ctggctcctc atatcggggg ggaggctggg
     3241 agctcacatg ccccgccccc ggccctcacc ctcatcttcg accgccatcc catcgccgcc
     3301 ctcctgtgct acccggccgc gcgatacctt atgggcagca tgacccccca ggccgtgctg
     3361 gcgttcgtgg ccctcatccc gccgaccttg cccggcacaa acatcgtgtt gggggccctt
     3421 ccggaggaca gacacatcga ccgcctggcc aaacgccagc gccccggcga gcggcttgac
     3481 ctggctatgc tggccgcgat tcgccgcgtt tacgggctgc ttgccaatac ggtgcggtat
     3541 ctgcagggcg gcgggtcgtg gcgggaggat tggggacagc tttcggggac ggccgtgccg
     3601 ccccagggtg ccgagcccca gagcaacgcg ggcccacgac cccatatcgg ggacacgtta
     3661 tttaccctgt ttcgggcccc cgagttgctg gcccccaacg gcgacctgta caacgtgttt
     3721 gcctgggcct tggacgtctt ggccaaacgc ctccgtccca tgcacgtctt tatcctggat
     3781 tacgaccaat cgcccgccgg ctgccgggac gccctgctgc aacttacctc cgggatggtc
     3841 cagacccacg tcaccacccc cggctccata ccgacgatct gcgacctggc gcgcacgttt
     3901 gcccgggaga tgggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg
     3961 cgctatgacg gcaataaaaa gacagaataa aacgcacggg tgttgggtcg tttgttcata
     4021 aacgcggaag ttcctattcc gaagttccta ttcttcaaat agtataggaa cttcacgggc
     4081 cagatatacg cgtcgaggcc ctttcgtctt cactcgagtt tactccctat cagtgataga
     4141 gaacgtatgt cgagtttact ccctatcagt gatagagaac gatgtcgagt ttactcccta
     4201 tcagtgatag agaacgtatg tcgagtttac tccctatcag tgatagagaa cgtatgtaga
     4261 gtttactccc tatcagtgat agagaacgta tgtcgagttt atccctatca gtgatagaga
     4321 acgtatgtcg agtttactcc ctatcagtga tagagaacgt atgtcgaggt aggcgtgtac
     4381 ggtgggaggc ctatataagc agagctcgtt tagtgaaccg tcagatcgcc tggaatgtct
     4441 agactggaca agagcaaagt cataaacggc gctctggaat tactcaatgg agtcggtatc
     4501 gaaggcctga cgacaaggaa actcgctcaa aagctgggag ttgagcagcc taccctgtac
     4561 tggcacgtga agaacaagcg ggccctgctc gatgccctgc caatcgagat gctggacagg
     4621 catcataccc acttctgccc cctggaaggc gagtcatggc aagactttct gcggaacaac
     4681 gccaagtcat tccgctgtgc tctcctctca catcgcgacg gggctaaagt gcatctcggc
     4741 acccgcccaa cagagaaaca gtacgaaacc ctggaaaatc agctcgcgtt cctgtgtcag
     4801 caaggcttct ccctggagaa cgcactgtac gctctgtccg ccgtgggcca ctttacactg
     4861 ggctgcgtat tggaggaaca ggagcatcaa gtagcaaaag aggaaagaga gacacctacc
     4921 accgattcta tgcccccact tctgagacaa gcaattgagc tgttcgaccg gcagggagcc
     4981 gaacctgcct tccttttcgg cctggaacta atcatatgtg gcctggagaa acagctaaag
     5041 tgcgaaagcg gcgggccggc cgacgccctt gacgattttg acttagacat gctcccagcc
     5101 gatgcccttg acgactttga ccttgatatg ctgcctgctg acgctcttga cgattttgac
     5161 cttgacatgc tccccggggg aagcggagct actaacttca gcctgctgaa gcaggctgga
     5221 gacgtggagg agaaccctgg acctatggtg agcaagggcg aggagctgtt caccggggtg
     5281 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     5341 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     5401 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     5461 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     5521 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     5581 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     5641 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     5701 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     5761 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     5821 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     5881 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     5941 ggcatggacg agctgtacaa gtaaggatcc actagtccag tgtggtggaa ttctgcagat
     6001 atccagcaca gtggcggccg ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct
     6061 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     6121 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     6181 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     6241 attgggaaga caatagcagg catgctgggg atgcggtggg ctc
//
