LOCUS       sequence-9956-ex        6292 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 6292)
  AUTHORS   Kaelin WG, Krek W, Sellers WR, DeCaprio JA, Ajchenbaum F, Fuchs CS, 
            Chittenden T, Li Y, Farnham PJ, Blanar MA
  TITLE     Expression cloning of a cDNA encoding a retinoblastoma-binding 
            protein with E2F-like properties.
  JOURNAL   Cell. 1992 Jul 24. 70(2):351-64.
  PUBMED    1638635
REFERENCE   2  (bases 1 to 6292)
  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. 1992 
            Jul 24. 70(2):351-64."
COMMENT     Sequence Label: E2F-1 wt-pGex2TK
FEATURES             Location/Qualifiers
     source          1..6292
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..211)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /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)."
     CDS             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             918..935
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(2342..2364)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2509..2527)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2595..2699
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2700..2768,2769..3560)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2918..2937)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3731..4319
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4220..4239
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4473..4490
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4563..4640
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             4641..5723
                     /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(4660..4679)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5736..5757
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5772..5777,5778..5795,5796..5802)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5810..5826
                     /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)."
     primer_bind     5834..5850
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5834..5850
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5846..6292,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGITLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5866..5883)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5866..5882)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5875..5897)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gttccgcgtg gatctcgtcg tgcatctgtt ggatcccata
      961 tggccttggc cggggcccct gcgggcggcc catgcgcgcc ggcgctggag gccctgctcg
     1021 gggccggcgc gctgcggctg ctcgactcct cgcagatcgt catcatctcc gccgcgcagg
     1081 acgccagcgc cccgccggct cccaccggcc ccgcggcgcc cgccgccggc ccctgcgacc
     1141 ctgacctgct gctcttcgcc acaccgcagg cgccccggcc cacacccagt gcgccgcggc
     1201 ccgcgctcgg ccgcccgccg gtgaagcgga ggctggacct ggaaactgac catcagtacc
     1261 tggccgagag cagtgggcca gctcggggca gaggccgcca tccaggaaaa ggtgtgaaat
     1321 ccccggggga gaagtcacgc tatgagacct cactgaatct gaccaccaag cgcttcctgg
     1381 agctgctgag ccactcggct gacggtgtcg tcgacctgaa ctgggctgcc gaggtgctga
     1441 aggtgcagaa gcggcgcatc tatgacatca ccaacgtcct tgagggcatc cagctcattg
     1501 ccaagaagtc caagaaccac atccagtggc tgggcagcca caccacagtg ggcgtcggcg
     1561 gacggcttga ggggttgacc caggacctcc gacagctgca ggagagcgag cagcagctgg
     1621 accacctgat gaatatctgt actacgcagc tgcgcctgct ctccgaggac actgacagcc
     1681 agcgcctggc ctacgtgacg tgtcaggacc ttcgtagcat tgcagaccct gcagagcaga
     1741 tggttatggt gatcaaagcc cctcctgaga cccagctcca agccgtggac tcttcggaga
     1801 actttcagat ctcccttaag agcaaacaag gcccgatcga tgttttcctg tgccctgagg
     1861 agaccgtagg tgggatcagc cctgggaaga ccccatccca ggaggtcact tctgaggagg
     1921 agaacagggc cactgactct gccaccatag tgtcaccacc accatcatct cccccctcat
     1981 ccctcaccac agatcccagc cagtctctac tcagcctgga gcaagaaccg ctgttgtccc
     2041 ggatgggcag cctgcgggct cccgtggacg aggaccgcct gtccccgctg gtggcggccg
     2101 actcgctcct ggagcatgtg cgggaggact tctccggcct cctccctgag gagttcatca
     2161 gcctttcccc accccacgag gccctcgact accacttcgg cctcgaggag ggcgagggca
     2221 tcagagacct cttcgactgt gactttgggg acctcacccc cctggatttc tgacaggatg
     2281 catgaattca tcgtgactga ctgacgatct gcctcgcgcg tttcggtgat gacggtgaaa
     2341 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     2401 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga
     2461 cccagtcacg tagcgatagc ggagtgtata attcttgaag acgaaagggc ctcgtgatac
     2521 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     2581 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     2641 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     2701 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     2761 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     2821 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     2881 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     2941 gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     3001 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     3061 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     3121 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     3181 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     3241 ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     3301 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     3361 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     3421 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     3481 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     3541 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     3601 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     3661 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     3721 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     3781 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     3841 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     3901 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     3961 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     4021 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     4081 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     4141 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     4201 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     4261 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     4321 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     4381 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     4441 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     4501 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcataaatt
     4561 ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga
     4621 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg
     4681 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa
     4741 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac
     4801 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc
     4861 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg
     4921 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc
     4981 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca
     5041 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga
     5101 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc
     5161 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg
     5221 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag
     5281 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga
     5341 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa
     5401 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg
     5461 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc
     5521 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
     5581 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
     5641 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     5701 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     5761 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     5821 taacaatttc acacaggaaa cagctatgac catgattacg gattcactgg ccgtcgtttt
     5881 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     5941 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     6001 gcgcagcctg aatggcgaat ggcgctttgc ctggtttccg gcaccagaag cggtgccgga
     6061 aagctggctg gagtgcgatc ttcctgaggc cgatactgtc gtcgtcccct caaactggca
     6121 gatgcacggt tacgatgcgc ccatctacac caacgtaacc tatcccatta cggtcaatcc
     6181 gccgtttgtt cccacggaga atccgacggg ttgttactcg ctcacattta atgttgatga
     6241 aagctggcta caggaaggcc agacgcgaat tatttttgat ggcgttggaa tt
//
