LOCUS       sequence-108250-        7463 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic autoregulatory gene circuit made by inserting an intron 
            containing the mouse mir-124–3 gene into mCherry. Contains the 
            miR-124-regulated 3′UTR of the Vamp3 gene with three siRNA-like 
            sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7463)
  AUTHORS   Strovas TJ, Rosenberg AB, Kuypers BE, Muscat RA, Seelig G
  TITLE     MicroRNA-based single-gene circuits buffer protein synthesis rates 
            against perturbations.
  JOURNAL   ACS Synth Biol. 2014 May 16;3(5):324-31. doi: 10.1021/sb4001867. 
            Epub 2014 Jan 30.
  PUBMED    24847681
REFERENCE   2  (bases 1 to 7463)
  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: "10.1021/sb4001867";
            journalName: "ACS Synth Biol"; date: "2014-05-16- 16"; volume: "3"; 
            issue: "5"; pages: "324-31"
COMMENT     Sequence Label: sgFFL_3x_siRNA
FEATURES             Location/Qualifiers
     source          1..7463
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             992..1369
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      1388..1397
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1539..1557)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1807..1826
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             complement(2642..2653)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     primer_bind     complement(3415..3432)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3421..3645
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3778..3797)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3929..3976
                     /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             3984..5006
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3984..5006
                     /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    5136..5257
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5173..5192)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5227..5246
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5306..5322)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5306..5322)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5319..5341)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5330..5346
                     /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(5354..5360,5361..5378,5379..5384))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5399..5420
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5537..5554)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5708..6296)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5788..5807)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6467..7258,7259..7327))
                     /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     7090..7109
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7328..7432)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag ctttcgccac catgccagag ccagcgaagt ctgctcccgc
     1021 cccgaaaaag ggctccaaga aggcggtgac taaggcgcag aagaaaggcg gcaagaagcg
     1081 caagcgcagc cgcaaggaga gctattccat ctatgtgtac aaggttctga agcaggtcca
     1141 ccctgacacc ggcatttcgt ccaaggccat gggcatcatg aattcgtttg tgaacgacat
     1201 tttcgagcgc atcgcaggtg aggcttcccg cctggcgcat tacaacaagc gctcgaccat
     1261 cacctccagg gagatccaga cggccgtgcg cctgctgctg cctggggagt tggccaagca
     1321 cgccgtgtcc gagggtacta aggccatcac caagtacacc agcgctaagg atccccgggt
     1381 accggtcgcc accatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt
     1441 catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg
     1501 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg
     1561 tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc
     1621 ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt
     1681 caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc
     1741 ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc
     1801 cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta
     1861 ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg
     1921 ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc aggtgagtac
     1981 tccctctcaa aagcgggcat gacttctgcg ctaagattgt cagtttccaa aaacgaggag
     2041 gatttgatat tcacctggcc cgcggtgatg cctttgaggg tggccgcgtc catctggtca
     2101 gaaaagacaa tctttttgtt gtcaagctgt ttgactcatg cacccgctac tcttcaacct
     2161 tgcagactaa ttaatggcaa ctctgacaga tgtggcggga ggaaagctga ggtgggagta
     2221 ctgctcagag ctacaactct aggagtaggg actccaagcc tagagctcca agagagggtg
     2281 aagggcaggg agaaaattat agtaatagtt gcaatgagtc acttgcttct agatcaagat
     2341 cagagactct gctctccgtg ttcacagcgg accttgattt aatgtcatac aattaaggca
     2401 cgcggtgaat gccaagagcg gagcctacgg ctgcacttga aggacatccg agagaagtta
     2461 ggaagggtgg ggagaaacaa ttctagaatg aacccatcct gtgcgacaca aggacccctc
     2521 gcattgattc aaaatcttta gaatacaaga cagtcaaatg tttttcagcg agaatctggg
     2581 aaatccgcat gcatactccc aaggggaata ttagaggttt cattgacagc tgtcaggaga
     2641 gctgtagttc ccattggtgc aaacggtcta agagatggca ctcagagagt gctggccttt
     2701 tccaaatact agtcatatgc ggccgcagct tgaggtgtga caggcttgag atctggccat
     2761 acacttgagt gacaatgaca tccactttgc ctttctctcc acagctgccc ggcgcctaca
     2821 acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt
     2881 acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac aagtctgacc
     2941 ttgatatcca gcacagtggc ggccgctcga gtagccatgg cttcccgccg gcggtggcgg
     3001 cgcaggatga tggcacgctg cccatgtctt gtgcccagga gagcgggatg gaccgtcacc
     3061 ctgcagcctg tgcttctgct aggatcaatg tgtagtaatg agtttaaggc gcgcggctct
     3121 agagcaccga tgaaactgaa gcccgatatt caggatcttt ctgaccgatt cgtgcttcgg
     3181 cattcaccgc gtgccttaca acagaggaca taagccctac gcccatgatc tactgacggg
     3241 cattcaccgc gtgccttagt aatgggacag tatccgcggc aagtcctagt gcaatggggc
     3301 attcaccgcg tgccttagta gtagaggcga cgcgggtgcg gtcatcacta ataaggatag
     3361 gaggtggatg ttgtcactca agatgacttg ttgtcgttta aacccgctga tcagcctcga
     3421 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     3481 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     3541 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     3601 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     3661 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
     3721 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     3781 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     3841 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     3901 ttgattaggg tgatggttca cgtacctaga agttcctatt ccgaagttcc tattctctag
     3961 aaagtatagg aacttccttg gccaaaaagc ctgaactcac cgcgacgtct gtcgagaagt
     4021 ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat
     4081 ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg
     4141 ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga
     4201 ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc
     4261 gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc
     4321 cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg
     4381 gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga
     4441 ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg
     4501 tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc
     4561 tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg
     4621 tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct
     4681 tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc
     4741 cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac
     4801 tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg
     4861 acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg
     4921 cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca
     4981 gcactcgtcc gagggcaaag gaatagcacg tactacgaga tttcgattcc accgccgcct
     5041 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     5101 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     5161 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     5221 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     5281 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     5341 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     5401 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5461 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5521 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5581 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     5641 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5701 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5761 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5821 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5881 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     5941 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     6001 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     6061 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     6121 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     6181 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6241 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6301 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6361 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6421 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6481 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6541 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6601 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     6661 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6721 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6781 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6841 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     6901 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6961 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     7021 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     7081 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     7141 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     7201 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     7261 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     7321 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     7381 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     7441 cccgaaaagt gccacctgac gtc
//
