LOCUS       sequence-9325-ex        4075 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4075)
  AUTHORS   Deans TL, Cantor CR, Collins JJ
  TITLE     A tunable genetic switch based on RNAi and repressor proteins for 
            regulating gene expression in mammalian cells.
  JOURNAL   Cell. 2007 Jul 27. 130(2):363-72.
  PUBMED    17662949
REFERENCE   2  (bases 1 to 4075)
  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: "Cell. 2007 
            Jul 27. 130(2):363-72."
COMMENT     Sequence Label: TLD_026
FEATURES             Location/Qualifiers
     source          1..4075
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        462..765
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        766..969
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     920..940
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1024..1644
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(1097..1116)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     polyA_signal    1686..1807
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1723..1742)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1777..1796
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1854..1870)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1854..1870)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1867..1889)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1878..1894
                     /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(1902..1908,1909..1926,1927..1932))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1947..1968
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2085..2102)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2256..2844)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2336..2355)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3015..3806,3807..3875))
                     /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     3638..3657
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3876..3980)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4048..4066
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ctagttatta atagtaatca
      421 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
      481 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
      541 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
      601 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
      661 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
      721 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
      781 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
      841 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
      901 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
      961 agcagagctg gtttagtgaa ccgtcagatc ggatcctcta gagtcgacct gcaggccgcc
     1021 accatgtcta gattagataa aagtaaagtg attaacagcg cattagagct gcttaatgag
     1081 gtcggaatcg aaggtttaac aacccgtaaa ctcgcccaga agctaggtgt agagcagcct
     1141 acattgtatt ggcatgtaaa aaataagcgg gctttgctcg acgccttagc cattgagatg
     1201 ttagataggc accatactca cttttgccct ttagaagggg aaagctggca agatttttta
     1261 cgtaataacg ctaaaagttt tagatgtgct ttactaagtc atcgcgatgg agcaaaagta
     1321 catttaggta cacggcctac agaaaaacag tatgaaactc tcgaaaatca attagccttt
     1381 ttatgccaac aaggtttttc actagagaat gcattatatg cactcagcgc tgtggggcat
     1441 tttactttag gttgcgtatt ggaagatcaa gagcatcaag tcgctaaaga agaaagggaa
     1501 acacctacta ctgatagtat gccgccatta ttacgacaag ctatcgaatt atttgatcac
     1561 caaggtgcag agccagcctt cttattcggc cttgaattga tcatatgcgg attagaaaaa
     1621 caacttaaat gtgaaagtgg gtccgcgtac agcggatccc gggaattcag atcttattaa
     1681 agcagaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     1741 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     1801 tatcttatca tgtctggtcg aatccatcac actggaagct tggcgtaatc atggtcatag
     1861 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     1921 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     1981 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     2041 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     2101 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     2161 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     2221 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     2281 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     2341 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     2401 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     2461 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     2521 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     2581 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     2641 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     2701 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     2761 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     2821 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     2881 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     2941 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     3001 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     3061 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     3121 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     3181 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     3241 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     3301 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     3361 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     3421 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     3481 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     3541 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     3601 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     3661 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     3721 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     3781 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     3841 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     3901 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     3961 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     4021 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtc
//
