LOCUS       sequence-251841-        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"
     promoter        complement(146..250)
                     /gene="bla"
                     /label=AmpR promoter
     regulatory      483..492
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      744..753
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1221..1241
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(1270..1287)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1276..1500
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        1697..2000
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2001..2204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2155..2175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             2908..2934
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3364..3735
                     /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             3736..3759
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    3880..4001
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4081..4100)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4232..4279
                     /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             4287..5309
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4287..5309
                     /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    5439..5560
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5609..5625)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5609..5625)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5622..5644)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5633..5649
                     /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(5657..5663,5664..5681,5682..5687))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5702..5723
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5840..5857)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6011..6599)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6091..6110)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6770..7485,1..76,77..145))
                     /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     7393..7412
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
       61 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
      121 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
      181 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
      241 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctcgataggt accgagtttc
      301 tagacggagt actgtcctcc gagcggagta ctgtcctccg actcgagcgg agtactgtcc
      361 tccgatcgga gtactgtcct ccgcgaattc cggagtactg tcctccgaag acgctagcgg
      421 ggggctataa aagggggtgg gggcgttcgt cctcactcta gatctgcgat ctaagtaagc
      481 ttgccaccat ggtgagcaag ggcgaggagg acaacatggc cagcctgccc gccacccacg
      541 agctgcacat cttcggcagc atcaacggcg tggacttcga catggtgggc cagggcaccg
      601 gcaaccccaa cgacggctac gaggagctga acctgaagag caccaagggc gacctgcagt
      661 tcagcccctg gatcctggtg ccccacatcg gctacggctt ccaccagtac ctgccctacc
      721 ccgacggcat gagccccttc caggccgcca tggtggacgg cagcggctac caggtgcaca
      781 ggaccatgca gttcgaggac ggcgccagcc tgaccgtgaa ctacaggtac acctacgagg
      841 gcagccacat caagggcgag gcccaggtga agggcaccgg cttccccgcc gacggccccg
      901 tgatgaccaa cagcctgacc gccgccgact ggtgcaggag caagaagacc taccccaacg
      961 acaagaccat catcagcacc ttcaagtgga gctacaccac cggcaacggc aagaggtaca
     1021 ggagcaccgc caggaccacc tacaccttcg ccaagcccat ggccgccaac tacctgaaga
     1081 accagcccat gtacgtgttc aggaagaccg agctgaagca cagcaagacc gagctgaact
     1141 tcaaggagtg gcagaaggcc ttcaccgacg tgatgggcat ggacgagctg tacaaggtta
     1201 acaagaagaa gcgcaaggtg cctaagaaga agaggaaggt gtaggggccc gtttaaaccc
     1261 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     1321 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     1381 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     1441 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     1501 cttctgaggc ggaaagaacc agctggggct ctagggggta tttttacggt tcctggcctt
     1561 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     1621 tattaccgcc atgcattagt tattaatagt aatcaattac ggggtcatta gttcatagcc
     1681 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
     1741 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
     1801 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
     1861 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
     1921 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
     1981 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
     2041 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
     2101 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
     2161 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
     2221 agatccgcta gcgctatgaa gctactgtct tctatcgaac aagcatgcga tatttgccga
     2281 cttaaaaagc tcaagtgctc caaagaaaaa ccgaagtgcg ccaagtgtct gaagaacaac
     2341 tgggagtgtc gctactctcc caaaaccaaa aggtctccgc tgactagggc acatctgaca
     2401 gaagtggaat caaggctaga aagactggaa agatccatcg ccaccagatc tcatacgctc
     2461 tacgctcccg gcggttatga cattatgggc tatctgattc agattatgaa gaggccaaac
     2521 ccccaagtag aactgggacc tgttgacacg tcagttgctc tgattctgtg cgacctgaag
     2581 caaaaagaca cgccaattgt gtacgcctcg gaagcttttc tctatatgac aggatacagc
     2641 aatgcggagg tcttggggag aaactgccgt tttcttcagt cacccgacgg aatggtcaag
     2701 ccgaaatcga caaggaagta cgtcgactcc aacacgatca atacgatgag gaaagcgatt
     2761 gataggaacg ccgaggtgca ggttgaggtg gtcaatttta agaagaacgg ccaacggttt
     2821 gtcaacttct tgacgatgat tccggtgcga gatgaaacag gggaataccg gtacagcatg
     2881 ggtttccagt gcgaaacgga actgcagtac ccatacgatg ttccagatta cgctgaattc
     2941 cagtacctgc cagatacaga cgatcgtcac cggattgagg agaaacgtaa aaggacatat
     3001 gagaccttca agagcatcat gaagaagagt cctttcagcg gacccaccga cccccggcct
     3061 ccacctcgac gcattgctgt gccttcccgc agctcagctt ctgtccccaa gccagcaccc
     3121 cagccctatc cctttacgtc atccctgagc accatcaact atgatgagtt tcccaccatg
     3181 gtgtttcctt ctgggcagat cagccaggcc tcggccttgg ccccggcccc tccccaagtc
     3241 ctgccccagg ctccagcccc tgcccctgct ccagccatgg tatcagctct ggcccaggcc
     3301 ccagcccctg tcccagtcct agccccaggc cctcctcagg ctgtggcccc acctgccccc
     3361 aagcccaccc aggctgggga aggaacgctg tcagaggccc tgctgcagct gcagtttgat
     3421 gatgaagacc tgggggcctt gcttggcaac agcacagacc cagctgtgtt cacagacctg
     3481 gcatccgtcg acaactccga gtttcagcag ctgctgaacc agggcatacc tgtggccccc
     3541 cacacaactg agcccatgct gatggagtac cctgaggcta taactcgcct agtgacaggg
     3601 gcccagaggc cccccgaccc agctcctgct ccactggggg ccccggggct ccccaatggc
     3661 ctcctttcag gagatgaaga cttctcctcc attgcggaca tggacttctc agccctgctg
     3721 agtcagatca gctccgacta caaagacgat gatgacaagt aatgtacact ctagatcata
     3781 atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc
     3841 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat
     3901 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     3961 cattctagtt gtggtttgtc caaactcatc aatgtatctt aagtagcggc gcattaagcg
     4021 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     4081 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     4141 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     4201 aacttgatta gggtgatggt tcacgtacct agaagttcct attccgaagt tcctattctc
     4261 tagaaagtat aggaacttcc ttggccaaaa agcctgaact caccgcgacg tctgtcgaga
     4321 agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag
     4381 aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct
     4441 gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc
     4501 cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc
     4561 gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc
     4621 agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt
     4681 tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg
     4741 cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt
     4801 ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc
     4861 acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag
     4921 cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct
     4981 tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc
     5041 atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc
     5101 aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat
     5161 gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa
     5221 gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc
     5281 ccagcactcg tccgagggca aaggaatagc acgtactacg agatttcgat tccaccgccg
     5341 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     5401 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     5461 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     5521 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     5581 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     5641 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     5701 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5761 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5821 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5881 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5941 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     6001 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     6061 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     6121 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     6181 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     6241 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     6301 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     6361 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     6421 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     6481 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6541 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6601 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6661 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6721 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6781 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6841 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6901 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6961 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     7021 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     7081 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     7141 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     7201 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     7261 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     7321 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     7381 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     7441 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttga
//
