LOCUS       sequence-9328-ex        4898 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 4898)
  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 4898)
  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_027
FEATURES             Location/Qualifiers
     source          1..4898
                     /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             1022..2101
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(1041..1060)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2117..2137
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          2299..2364
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     polyA_signal    2523..2644
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2560..2579)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2614..2633
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2677..2693)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2677..2693)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2690..2712)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2701..2717
                     /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(2725..2731,2732..2749,2750..2755))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2770..2791
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2908..2925)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3079..3667)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3159..3178)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3838..4629,4630..4698))
                     /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     4461..4480
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4699..4803)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4871..4889
                     /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 gagtcgacca ttgccgccac
     1021 catgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt
     1081 ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc
     1141 ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca
     1201 gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt
     1261 cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga
     1321 acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag
     1381 tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg
     1441 cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat
     1501 tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca
     1561 gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg
     1621 ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg
     1681 gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac
     1741 tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc
     1801 cgggctgcgc gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc
     1861 atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag
     1921 cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc
     1981 cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg
     2041 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     2101 gagcagcctg aggcctccta agaagaagag gaaggtttga gcgcaacgca attaatgtaa
     2161 gttagctcac tcattaggca ccccaggctt tacactttat gcttccgacc tgcagcccct
     2221 tggatctttg tgaaggaacc ttacttctgt ggtgtgacat aattggacaa actacctaca
     2281 gagatttaaa gctctaaggt aaatataaaa tttttaagtg tataatgtgt taaactactg
     2341 attctaattg tttgtgtatt ttagatcaca gtcccaaggc tcatttcagg cccctcagtc
     2401 ctcacagtct gttcatgatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt
     2461 taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg
     2521 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     2581 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     2641 cttatcatgt ctgggcccaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     2701 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     2761 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     2821 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     2881 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     2941 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     3001 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     3061 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     3121 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     3181 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     3241 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     3301 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     3361 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     3421 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     3481 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     3541 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     3601 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     3661 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     3721 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     3781 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     3841 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     3901 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     3961 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     4021 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     4081 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     4141 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     4201 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     4261 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     4321 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     4381 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     4441 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     4501 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     4561 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     4621 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     4681 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     4741 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     4801 gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt
     4861 aacctataaa aataggcgta tcacgaggcc ctttcgtc
//
