LOCUS       sequence-245705-        7485 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gene circuit with the following architecture: 
            5xUAS-mNeonGreen--CMV-Gal4-VVD-p65.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7485)
  AUTHORS   Guinn MT, Balazsi G
  TITLE     Noise-reducing optogenetic negative-feedback gene circuits in human 
            cells.
  JOURNAL   Nucleic Acids Res. 2019 Jul 3. pii: 5527975. doi: 
            10.1093/nar/gkz556.
  PUBMED    31269201
REFERENCE   2  (bases 1 to 7485)
  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"; journalName: "Nucleic 
            Acids Res. 2019 Jul 3. pii: 5527975. doi: 10.1093/nar/gkz556."
COMMENT     Sequence Label: pMTG07-Amp-FRT
FEATURES             Location/Qualifiers
     source          1..7485
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(128..716)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(208..227)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(887..1678,1679..1747))
                     /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     1510..1529
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1748..1852)
                     /gene="bla"
                     /label=AmpR promoter
     regulatory      2085..2094
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2346..2355
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2823..2843
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2872..2889)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2878..3102
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        3299..3602
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3603..3806
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3757..3777
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             4510..4536
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4966..5337
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALLSQISS"
     CDS             5338..5361
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    5482..5603
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5683..5702)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    5834..5881
                     /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             5889..6911
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             5889..6911
                     /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    7041..7162
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7211..7227)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7211..7227)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7224..7246)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7235..7251
                     /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(7259..7265,7266..7283,7284..7289))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7304..7325
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7442..7459)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
       61 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
      121 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
      181 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
      241 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
      301 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
      361 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
      421 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
      481 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
      541 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
      601 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
      661 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
      721 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
      781 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
      841 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
      901 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
      961 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     1021 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     1081 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     1141 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     1201 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     1261 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     1321 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     1381 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     1441 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     1501 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     1561 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     1621 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     1681 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     1741 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     1801 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     1861 cccgaaaagt gccacctgac gtctcgatag gtaccgagtt tctagacgga gtactgtcct
     1921 ccgagcggag tactgtcctc cgactcgagc ggagtactgt cctccgatcg gagtactgtc
     1981 ctccgcgaat tccggagtac tgtcctccga agacgctagc ggggggctat aaaagggggt
     2041 gggggcgttc gtcctcactc tagatctgcg atctaagtaa gcttgccacc atggtgagca
     2101 agggcgagga ggacaacatg gccagcctgc ccgccaccca cgagctgcac atcttcggca
     2161 gcatcaacgg cgtggacttc gacatggtgg gccagggcac cggcaacccc aacgacggct
     2221 acgaggagct gaacctgaag agcaccaagg gcgacctgca gttcagcccc tggatcctgg
     2281 tgccccacat cggctacggc ttccaccagt acctgcccta ccccgacggc atgagcccct
     2341 tccaggccgc catggtggac ggcagcggct accaggtgca caggaccatg cagttcgagg
     2401 acggcgccag cctgaccgtg aactacaggt acacctacga gggcagccac atcaagggcg
     2461 aggcccaggt gaagggcacc ggcttccccg ccgacggccc cgtgatgacc aacagcctga
     2521 ccgccgccga ctggtgcagg agcaagaaga cctaccccaa cgacaagacc atcatcagca
     2581 ccttcaagtg gagctacacc accggcaacg gcaagaggta caggagcacc gccaggacca
     2641 cctacacctt cgccaagccc atggccgcca actacctgaa gaaccagccc atgtacgtgt
     2701 tcaggaagac cgagctgaag cacagcaaga ccgagctgaa cttcaaggag tggcagaagg
     2761 ccttcaccga cgtgatgggc atggacgagc tgtacaaggt taacaagaag aagcgcaagg
     2821 tgcctaagaa gaagaggaag gtgtaggggc ccgtttaaac ccgctgatca gcctcgactg
     2881 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     2941 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     3001 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     3061 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     3121 ccagctgggg ctctaggggg tatttttacg gttcctggcc ttttgctggc cttttgctca
     3181 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg ccatgcatta
     3241 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
     3301 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     3361 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     3421 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
     3481 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
     3541 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     3601 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
     3661 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
     3721 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
     3781 ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagatccgc tagcgctatg
     3841 aagctactgt cttctatcga acaagcatgc gatatttgcc gacttaaaaa gctcaagtgc
     3901 tccaaagaaa aaccgaagtg cgccaagtgt ctgaagaaca actgggagtg tcgctactct
     3961 cccaaaacca aaaggtctcc gctgactagg gcacatctga cagaagtgga atcaaggcta
     4021 gaaagactgg aaagatccat cgccaccaga tctcatacgc tctacgctcc cggcggttat
     4081 gacattatgg gctatctgat tcagattatg aagaggccaa acccccaagt agaactggga
     4141 cctgttgaca cgtcagttgc tctgattctg tgcgacctga agcaaaaaga cacgccaatt
     4201 gtgtacgcct cggaagcttt tctctatatg acaggataca gcaatgcgga ggtcttgggg
     4261 agaaactgcc gttttcttca gtcacccgac ggaatggtca agccgaaatc gacaaggaag
     4321 tacgtcgact ccaacacgat caatacgatg aggaaagcga ttgataggaa cgccgaggtg
     4381 caggttgagg tggtcaattt taagaagaac ggccaacggt ttgtcaactt cttgacgatg
     4441 attccggtgc gagatgaaac aggggaatac cggtacagca tgggtttcca gtgcgaaacg
     4501 gaactgcagt acccatacga tgttccagat tacgctgaat tccagtacct gccagataca
     4561 gacgatcgtc accggattga ggagaaacgt aaaaggacat atgagacctt caagagcatc
     4621 atgaagaaga gtcctttcag cggacccacc gacccccggc ctccacctcg acgcattgct
     4681 gtgccttccc gcagctcagc ttctgtcccc aagccagcac cccagcccta tccctttacg
     4741 tcatccctga gcaccatcaa ctatgatgag tttcccacca tggtgtttcc ttctgggcag
     4801 atcagccagg cctcggcctt ggccccggcc cctccccaag tcctgcccca ggctccagcc
     4861 cctgcccctg ctccagccat ggtatcagct ctggcccagg ccccagcccc tgtcccagtc
     4921 ctagccccag gccctcctca ggctgtggcc ccacctgccc ccaagcccac ccaggctggg
     4981 gaaggaacgc tgtcagaggc cctgctgcag ctgcagtttg atgatgaaga cctgggggcc
     5041 ttgcttggca acagcacaga cccagctgtg ttcacagacc tggcatccgt cgacaactcc
     5101 gagtttcagc agctgctgaa ccagggcata cctgtggccc cccacacaac tgagcccatg
     5161 ctgatggagt accctgaggc tataactcgc ctagtgacag gggcccagag gccccccgac
     5221 ccagctcctg ctccactggg ggccccgggg ctccccaatg gcctcctttc aggagatgaa
     5281 gacttctcct ccattgcgga catggacttc tcagccctgc tgagtcagat cagctccgac
     5341 tacaaagacg atgatgacaa gtaatgtaca ctctagatca taatcagcca taccacattt
     5401 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     5461 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc
     5521 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     5581 tccaaactca tcaatgtatc ttaagtagcg gcgcattaag cgcggcgggt gtggtggtta
     5641 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     5701 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     5761 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     5821 gttcacgtac ctagaagttc ctattccgaa gttcctattc tctagaaagt ataggaactt
     5881 ccttggccaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt
     5941 tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct
     6001 tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca
     6061 aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg
     6121 acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca
     6181 cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca
     6241 tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc
     6301 aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg
     6361 tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg
     6421 atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt
     6481 tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg
     6541 aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt
     6601 tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat
     6661 cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg
     6721 ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat
     6781 ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg
     6841 atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg
     6901 caaaggaata gcacgtacta cgagatttcg attccaccgc cgccttctat gaaaggttgg
     6961 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     7021 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     7081 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     7141 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     7201 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     7261 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     7321 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     7381 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     7441 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgag
//
