LOCUS       sequence-9327-ex        4344 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 4344)
  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 4344)
  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_010
FEATURES             Location/Qualifiers
     source          1..4344
                     /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        426..805
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        806..1009
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     960..980
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    1011..1029
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1032..1050
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1060..1084
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(1181..1183,1184..1186,1187..1900)
                     /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(1181..1183,1184..1186,1187..1897)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1226..1247)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1487..1506)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1834..1855
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2023..2104
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2060..2079)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2123..2139)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2123..2139)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2136..2158)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2147..2163
                     /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(2171..2177,2178..2195,2196..2201))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2216..2237
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2354..2371)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2525..3113)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2605..2624)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3284..4075,4076..4144))
                     /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     3907..3926
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4145..4249)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4317..4335
                     /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 catgtacggg ccagatatac
      421 gcgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca
      481 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
      541 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
      601 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
      661 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
      721 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
      781 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg
      841 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
      901 gttttggaac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac
      961 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc tccctatcag
     1021 tgatagagat ctccctatca gtgatagaga tcgtcgacga gctcgtttag tgaaccgtca
     1081 gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc
     1141 cagcctccgg gatcctctag agtcgacctg cagcacaacc atggtgagca agggcgagga
     1201 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     1261 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     1321 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     1381 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     1441 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     1501 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     1561 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     1621 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     1681 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     1741 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     1801 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     1861 cgccgggatc actctcggca tggacgagct gtacaagtaa agcggccgcg actctagatc
     1921 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
     1981 cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct
     2041 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     2101 ctgcaagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     2161 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     2221 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     2281 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     2341 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     2401 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     2461 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     2521 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     2581 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     2641 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     2701 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     2761 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     2821 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     2881 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     2941 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     3001 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     3061 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     3121 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     3181 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     3241 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     3301 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     3361 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     3421 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     3481 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     3541 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     3601 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     3661 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     3721 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     3781 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     3841 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     3901 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     3961 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     4021 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     4081 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     4141 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     4201 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     4261 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata
     4321 ggcgtatcac gaggcccttt cgtc
//
