LOCUS       sequence-396466-        7685 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 7685)
  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 7685)
  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..7685
                     /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           3342..3381
                     /label=HSV TK 5'-UTR
                     /note="5' untranslated region from the herpes simplex virus
                     thymidine kinase gene"
     CDS             3399..4529
                     /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(4527..4546)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4571..4619
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    4643..4676
                     /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        4697..5076
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5077..5280
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     protein_bind    5276..5294
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5302..5320
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     intron          5503..6075
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     regulatory      6087..6096
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6744..6800
                     /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(6801..6803,6804..6806,6807..7520)
                     /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(6801..6803,6804..6806,6807..7517)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6846..6867)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(7107..7126)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(7579..7599)
                     /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 acccgcttaa cagcgtcaac agcgtgccgc agatcttggt ggcgtgaaac
     3361 tcccgcacct cttcggcaag cgccttgtag aagcgcgtat ggcttcgtac ccctgccatc
     3421 aacacgcgtc tgcgttcgac caggctgcgc gttctcgcgg ccatagcaac cgacgtacgg
     3481 cgttgcgccc tcgccggcag caagaagcca cggaagtccg cctggagcag aaaatgccca
     3541 cgctactgcg ggtttatata gacggtcctc acgggatggg gaaaaccacc accacgcaac
     3601 tgctggtggc cctgggttcg cgcgacgata tcgtctacgt acccgagccg atgacttact
     3661 ggcaggtgct gggggcttcc gagacaatcg cgaacatcta caccacacaa caccgcctcg
     3721 accagggtga gatatcggcc ggggacgcgg cggtggtaat gacaagcgcc cagataacaa
     3781 tgggcatgcc ttatgccgtg accgacgccg ttctggctcc tcatatcggg ggggaggctg
     3841 ggagctcaca tgccccgccc ccggccctca ccctcatctt cgaccgccat cccatcgccg
     3901 ccctcctgtg ctacccggcc gcgcgatacc ttatgggcag catgaccccc caggccgtgc
     3961 tggcgttcgt ggccctcatc ccgccgacct tgcccggcac aaacatcgtg ttgggggccc
     4021 ttccggagga cagacacatc gaccgcctgg ccaaacgcca gcgccccggc gagcggcttg
     4081 acctggctat gctggccgcg attcgccgcg tttacgggct gcttgccaat acggtgcggt
     4141 atctgcaggg cggcgggtcg tggcgggagg attggggaca gctttcgggg acggccgtgc
     4201 cgccccaggg tgccgagccc cagagcaacg cgggcccacg accccatatc ggggacacgt
     4261 tatttaccct gtttcgggcc cccgagttgc tggcccccaa cggcgacctg tacaacgtgt
     4321 ttgcctgggc cttggacgtc ttggccaaac gcctccgtcc catgcacgtc tttatcctgg
     4381 attacgacca atcgcccgcc ggctgccggg acgccctgct gcaacttacc tccgggatgg
     4441 tccagaccca cgtcaccacc cccggctcca taccgacgat ctgcgacctg gcgcgcacgt
     4501 ttgcccggga gatgggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc
     4561 cgcgctatga cggcaataaa aagacagaat aaaacgcacg ggtgttgggt cgtttgttca
     4621 taaacgcgga agttcctatt ccgaagttcc tattcttcaa atagtatagg aacttcacgg
     4681 gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg
     4741 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
     4801 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
     4861 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     4921 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
     4981 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
     5041 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
     5101 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
     5161 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
     5221 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcatccct
     5281 atcagtgata gagatcagat ctccctatca gtgatagaga gctgttttga cctccataga
     5341 agacaccggg accgatccag cctccggact ctagcgttta aacttaagct tggtacccgg
     5401 ggatcctcta gggcctctga gctattccag aagtagtgaa gaggcttttt tggaggccta
     5461 ggcttttgca aaaagctccg gatcgatcct gagaacttca gggtgagttt ggggaccctt
     5521 gattgttctt tctttttcgc tattgtaaaa ttcatgttat atggaggggg caaagttttc
     5581 agggtgttgt ttagaatggg aagatgtccc ttgtatcacc atggaccctc atgataattt
     5641 tgtttctttc actttctact ctgttgacaa ccattgtctc ctcttatttt cttttcattt
     5701 tctgtaactt tttcgttaaa ctttagcttg catttgtaac gaatttttaa attcactttt
     5761 gtttatttgt cagattgtaa gtactttctc taatcacttt tttttcaagg caatcagggt
     5821 atattatatt gtacttcagc acagttttag agaacaattg ttataattaa atgataaggt
     5881 agaatatttc tgcatataaa ttctggctgg cgtggaaata ttcttattgg tagaaacaac
     5941 tacatcctgg tcatcatcct gcctttctct ttatggttac aatgatatac actgtttgag
     6001 atgaggataa aatactctga gtccaaaccg ggcccctctg ctaaccatgt tcatgccttc
     6061 ttctttttcc tacaggtcct gcaggcgcca ccatgggttc tagactggac aagagcaaag
     6121 tcataaactc tgctctggaa ttactcaatg aagtcggtat cgaaggcctg acgacaagga
     6181 aactcgctca aaagctggga gttgagcagc ctaccctgta ctggcacgtg aagaacaagc
     6241 gggccctgct cgatgccctg gcaatcgaga tgctggacag gcatcatacc cacttctgcc
     6301 ccctggaagg cgagtcatgg caagactttc tgcggaacaa cgccaagtca ttccgctgtg
     6361 ctctcctctc acatcgcgac ggggctaaag tgcatctcgg cacccgccca acagagaaac
     6421 agtacgaaac cctggaaaat cagctcgcgt tcctgtgtca gcaaggcttc tccctggaga
     6481 acgcactgta cgctctgtcc gccgtgggcc actttacact gggctgcgta ttggaggatc
     6541 aggagcatca agtagcaaaa gaggaaagag agacacctac caccgattct atgcccccac
     6601 ttctgagaca agcaattgag ctgttcgacc atcagggagc cgaacctgcc ttccttttcg
     6661 gcctggaact aatcatatgt ggcctggaga aacagctaaa gtgcgaaagc ggcgggccaa
     6721 aaaagaagag aaagggaagc ggagctacta acttcagcct gctgaagcag gctggagacg
     6781 tggaggagaa ccctggacct atggtgagca agggcgagga gctgttcacc ggggtggtgc
     6841 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
     6901 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     6961 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
     7021 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     7081 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     7141 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     7201 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     7261 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     7321 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     7381 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
     7441 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     7501 tggacgagct gtacaagtaa gccgcaatca acctctggat tacaaaattt gtgaaagatt
     7561 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     7621 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     7681 gttgc
//
