LOCUS       sequence-391952-        7684 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  LPUtopia-7 matching RMCE donor plasmid with GFP Negative-Feedback 
            circuit (mNF-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 7684)
  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 7684)
  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-mNF-GFP
FEATURES             Location/Qualifiers
     source          1..7684
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(267..284)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    273..497
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    632..1026
                     /label=beta-globin poly(A)
                     /note="human beta-globin polyadenylation signal"
     promoter        complement(1467..1485)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1477..1523)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(1570..1772)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     enhancer        1774..2077
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    2121..2168
                     /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(2180..2768)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2260..2279)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2801..3300
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2836..2859)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3233..3252
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     5'UTR           3341..3380
                     /label=HSV TK 5'-UTR
                     /note="5' untranslated region from the herpes simplex virus
                     thymidine kinase gene"
     CDS             3398..4528
                     /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(4526..4545)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4570..4618
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    4642..4675
                     /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)"
     enhancer        4696..5075
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5076..5279
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     protein_bind    5275..5293
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5301..5319
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     intron          5502..6074
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     regulatory      6086..6095
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6743..6799
                     /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(6800..6802,6803..6805,6806..7519)
                     /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(6800..6802,6803..6805,6806..7516)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6845..6866)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(7106..7125)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(7578..7598)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
ORIGIN
        1 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
       61 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
      121 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
      181 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcc tcgagtctag
      241 agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
      301 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
      361 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
      421 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
      481 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
      541 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gctaaagcaa
      601 gtaaaacctc tacaaatgtg gtatggaatt ctaaaataca gcatagcaaa actttaacct
      661 ccaaatcaag cctctacttg aatccttttc tgagggatga ataaggcata ggcatcaggg
      721 gctgttgcca atgtgcatta gctgtttgca gcctcacctt ctttcatgga gtttaagata
      781 tagtgtattt tcccaaggtt tgaactagct cttcatttct ttatgtttta aatgcactga
      841 cctcccacat tcccttttta gtaaaatatt cagaaataat ttaaatacat cattgcaatg
      901 aaaataaatg ttttttatta ggcagaatcc agatgctcaa ggcccttcat aatatccccc
      961 agtttagtag ttggacttag ggaacaaagg aacctttaat agaaattgga cagcaagaaa
     1021 gcgagcttct agctttagtt cctggtgtac ttgaggggga tgagttcctc aatggtggtt
     1081 ttgaccagct tgccattcat ctcaatgagc acaaagcagt caggagcata gtcagagatg
     1141 agctctctgc acatgccaca ggggctgacc accctgatgg atctgtccac ctcatcagag
     1201 taggggtgcc tgacagccac aatggtgtca aagtccttct gcccgttgct cacagcagac
     1261 ccaatggcaa tggcttcagc acagacagtg accctgccaa tgtaggcctc aatgtggaca
     1321 gcagagatga tctccccagt cttggtcctg atggccgccc cgacatggtg cttgttgtcc
     1381 tcatagagca tggtgatctt ctcagtggcg acctccacca gctccagatc ctgctgagag
     1441 atgttgaagg tcttcatggt ggccctccta tagtgagtcg tattatacta tgccgatata
     1501 ctatgccgat gattaattgt caaaacagcg tggatggcgt ctccagctta tctgacggtt
     1561 cactaaacga gctctgctta tatagacctc ccaccgtaca cgcctaccgc ccatttgcgt
     1621 caatggggcg gagttgttac gacattttgg aaagtcccgt tgatttacta gtcaaaacaa
     1681 actcccattg acgtcaatgg ggtggagact tggaaatccc cgtgagtcaa accgctatcc
     1741 acgcccattg atgtactgcc aaaaccgcat catcatggta atagcgatga ctaatacgta
     1801 gatgtactgc caagtaggaa agtcccataa ggtcatgtac tgggcataat gccaggcggg
     1861 ccatttaccg tcattgacgt caataggggg cgtacttggc atatgataca cttgatgtac
     1921 tgccaagtgg gcagtttacc gtaaatactc cacccattga cgtcaatgga aagtccctat
     1981 tggcgttact atgggaacat acgtcattat tgacgtcaat gggcgggggt cgttgggcgg
     2041 tcagccaggc gggccattta ccgtaagtta tgtaacgcct gcaggttaat taagaacatg
     2101 tgagcaaaag gccagcaaaa gaagttccta ttccgaagtt cctattctct agaaagtata
     2161 ggaacttctt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2221 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2281 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     2341 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     2401 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     2461 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     2521 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     2581 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     2641 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     2701 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     2761 gatctcaaga agatcctttg atcttttcta cggttctacc gggtagggga ggcgcttttc
     2821 ccaaggcagt ctggagcatg cgctttagca gccccgctgg gcacttggcg ctacacaagt
     2881 ggcctctggc ctcgcacaca ttccacatcc accggtaggc gccaaccggc tccgttcttt
     2941 ggtggcccct tcgcgccacc ttctactcct cccctagtca ggaagttccc ccccgccccg
     3001 cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac gtctcactag tctcgtgcag
     3061 atggacagca ccgctgagca atggaagcgg gtaggccttt ggggcagcgg ccaatagcag
     3121 ctttgctcct tcgctttctg ggctcagagg ctgggaaggg gtgggtccgg gggcgggctc
     3181 aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct ccggaggccc ggcattctgc
     3241 acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt cctcatctcc gggcctttcg
     3301 acctgcagcg acccgctaac agcgtcaaca gcgtgccgca gatcttggtg gcgtgaaact
     3361 cccgcacctc ttcggccagc gccttgtaga agcgcgtatg gcttcgtacc cctgccatca
     3421 acacgcgtct gcgttcgacc aggctgcgcg ttctcgcggc cataacaacc gacgtacggc
     3481 gttgcgccct cgccggcaac aaaaagccac ggaagtccgc ctggagcaga aaatgcccac
     3541 gctactgcgg gtttatatag acggtcccca cgggatgggg aaaaccacca ccacgcaact
     3601 gctggtggcc ctgggttcgc gcgacgatat cgtctacgta cccgagccga tgacttactg
     3661 gcgggtgttg ggggcttccg agacaatcgc gaacatctac accacacaac accgcctcga
     3721 ccagggtgag atatcggccg gggacgcggc ggtggtaatg acaagcgccc agataacaat
     3781 gggcatgcct tatgccgtga ccgacgccgt tctggctcct catatcgggg gggaggctgg
     3841 gagctcacat gccccgcccc cggccctcac cctcatcttc gaccgccatc ccatcgccgc
     3901 cctcctgtgc tacccggccg cgcgatacct tatgggcagc atgacccccc aggccgtgct
     3961 ggcgttcgtg gccctcatcc cgccgacctt gcccggcaca aacatcgtgt tgggggccct
     4021 tccggaggac agacacatcg accgcctggc caaacgccag cgccccggcg agcggcttga
     4081 cctggctatg ctggccgcga ttcgccgcgt ttatgggctg cttgccaata cggtgcggta
     4141 tctgcagggc ggcgggtcgt ggcgggagga ttggggacag ctttcggggg cggccgtgcc
     4201 gccccagggt gccgagcccc agagcaacgc gggcccacga ccccatatcg gggacacgtt
     4261 atttaccctg tttcgggccc ccgagttgct ggcccccaac ggcgacctgt acaacgtgtt
     4321 tgcctgggcc ttggacgtct tggccaaacg cctccgtccc atgcacgtct ttatcctgga
     4381 ttacgaccaa tcgcccgccg gctgccggga cgccctgctg caacttacct ccgggatggt
     4441 ccagacccac gtcaccaccc ccggctccat accgacgatc tgcgacctgg cgcgcacgtt
     4501 tgcccgggag atgggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc
     4561 gcgctatgac ggcaataaaa agacagaata aaacgcacgg gtgttgggtc gtttgttcat
     4621 aaacgcggaa gttcctattc cgaagttcct attcttcaaa tagtatagga acttcacggg
     4681 ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt
     4741 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     4801 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     4861 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     4921 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     4981 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     5041 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
     5101 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
     5161 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
     5221 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcatcccta
     5281 tcagtgatag agatcagatc tccctatcag tgatagagag ctgttttgac ctccatagaa
     5341 gacaccggga ccgatccagc ctccggactc tagcgtttaa acttaagctt ggtacccggg
     5401 gatcctctag ggcctctgag ctattccaga agtagtgaag aggctttttt ggaggcctag
     5461 gcttttgcaa aaagctccgg atcgatcctg agaacttcag ggtgagtttg gggacccttg
     5521 attgttcttt ctttttcgct attgtaaaat tcatgttata tggagggggc aaagttttca
     5581 gggtgttgtt tagaatggga agatgtccct tgtatcacca tggaccctca tgataatttt
     5641 gtttctttca ctttctactc tgttgacaac cattgtctcc tcttattttc ttttcatttt
     5701 ctgtaacttt ttcgttaaac tttagcttgc atttgtaacg aatttttaaa ttcacttttg
     5761 tttatttgtc agattgtaag tactttctct aatcactttt ttttcaaggc aatcagggta
     5821 tattatattg tacttcagca cagttttaga gaacaattgt tataattaaa tgataaggta
     5881 gaatatttct gcatataaat tctggctggc gtggaaatat tcttattggt agaaacaact
     5941 acatcctggt catcatcctg cctttctctt tatggttaca atgatataca ctgtttgaga
     6001 tgaggataaa atactctgag tccaaaccgg gcccctctgc taaccatgtt catgccttct
     6061 tctttttcct acaggtcctg caggcgccac catgggttct agactggaca agagcaaagt
     6121 cataaactct gctctggaat tactcaatga agtcggtatc gaaggcctga cgacaaggaa
     6181 actcgctcaa aagctgggag ttgagcagcc taccctgtac tggcacgtga agaacaagcg
     6241 ggccctgctc gatgccctgg caatcgagat gctggacagg catcataccc acttctgccc
     6301 cctggaaggc gagtcatggc aagactttct gcggaacaac gccaagtcat tccgctgtgc
     6361 tctcctctca catcgcgacg gggctaaagt gcatctcggc acccgcccaa cagagaaaca
     6421 gtacgaaacc ctggaaaatc agctcgcgtt cctgtgtcag caaggcttct ccctggagaa
     6481 cgcactgtac gctctgtccg ccgtgggcca ctttacactg ggctgcgtat tggaggatca
     6541 ggagcatcaa gtagcaaaag aggaaagaga gacacctacc accgattcta tgcccccact
     6601 tctgagacaa gcaattgagc tgttcgacca tcagggagcc gaacctgcct tccttttcgg
     6661 cctggaacta atcatatgtg gcctggagaa acagctaaag tgcgaaagcg gcgggccaaa
     6721 aaagaagaga aagggaagcg gagctactaa cttcagcctg ctgaagcagg ctggagacgt
     6781 ggaggagaac cctggaccta tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     6841 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     6901 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     6961 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     7021 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     7081 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     7141 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     7201 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     7261 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     7321 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     7381 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     7441 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     7501 ggacgagctg tacaagtaag ccgcaatcaa cctctggatt acaaaatttg tgaaagattg
     7561 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     7621 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
     7681 ttgc
//
