LOCUS       sequence-8428-ex        4609 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 4609)
  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 4609)
  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: Transgene Module (TrZ)
FEATURES             Location/Qualifiers
     source          1..4609
                     /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"
     promoter        663..924
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     protein_bind    998..1017
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     protein_bind    1041..1060
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     protein_bind    1084..1103
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     CDS             join(1459..1461,1462..1464,1465..2178)
                     /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(1459..1461,1462..1464,1465..2175)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1504..1525)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1765..1784)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2112..2133
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2325..2344)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2388..2404)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2388..2404)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2401..2423)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2412..2428
                     /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(2436..2442,2443..2460,2461..2466))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2481..2502
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2619..2636)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2790..3378)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2870..2889)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3549..4340,4341..4409))
                     /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     4172..4191
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4410..4514)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4582..4600
                     /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 atcctgctcc ctgcttgtgt
      421 gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac
      481 cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg
      541 ggccagatat acgcgtatct gaggggacta gggtgtgttt aggcgaaaag cggggcttcg
      601 gttgtacgcg gttaggagtc ccctcaggat atagtagttt cgcttttgca tagggagggg
      661 gaaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
      721 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
      781 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
      841 ttccgcattg cagagatatt gtatttaagt gcctagctcg atacaataaa cgccatttga
      901 ccattcacca cattggtgtg caccagatct aagcttggac aaactaccta cagagattta
      961 aagctctaag gtaaatataa aatttactag gttgtggaat tgtgagcgct cacaattcca
     1021 cagtcgaccc taggttgtgg aattgtgagc gctcacaatt ccacagtcga ccctaggttg
     1081 tggaattgtg agcgctcaca attccacagt cgaccctagt gtataatgtg ttaaactacg
     1141 gatccgtctc ccattaggcc tacaatggtg agacaagtag ccaacaggga agggttgcaa
     1201 atatcatttg ggcacaccta tgataatatt gatgaagcag acagtattca gcaagtaact
     1261 gagaggtggg aagctcaaag ccaaagtcct aatgtgcagt caggtgaatt tattgaaaaa
     1321 tttgaggctc ctggtggtgc aaatcaaaga actgctcctc agggatccta attgtttgtg
     1381 tattttagat tccaaccaag cttgcggccg ctcaggagct aaggaattga tcctctagag
     1441 tcgacctgca gcacaaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     1501 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     1561 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     1621 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     1681 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     1741 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     1801 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     1861 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     1921 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     1981 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     2041 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
     2101 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     2161 gacgagctgt acaagtaaag cggccgcgac tctagatcat aatcagccat accacatttg
     2221 tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa
     2281 tgaatgcact acacaaatca gcgattttaa tggttacaaa taaagcaata gcatcacaaa
     2341 tttcacaaat aaagcatttt tttcactgca agcttggcgt aatcatggtc atagctgttt
     2401 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     2461 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     2521 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     2581 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     2641 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     2701 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     2761 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     2821 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     2881 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     2941 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     3001 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     3061 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     3121 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     3181 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     3241 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     3301 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     3361 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     3421 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     3481 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     3541 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     3601 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     3661 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     3721 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     3781 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     3841 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     3901 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     3961 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     4021 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     4081 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     4141 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     4201 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     4261 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     4321 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     4381 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     4441 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     4501 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     4561 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtc
//
