LOCUS       sequence-9982-ex        6639 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 6639)
  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 6639)
  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: Rb-379-928-pGex2TK
FEATURES             Location/Qualifiers
     source          1..6639
                     /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(2689..2711)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2856..2874)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2942..3046
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3047..3115,3116..3907)
                     /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(3265..3284)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4078..4666
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4567..4586
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4820..4837
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4910..4987
                     /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             4988..6070
                     /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(5007..5026)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    6083..6104
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6119..6124,6125..6142,6143..6149)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6157..6173
                     /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     6181..6197
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6181..6197
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(6193..6639,1..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(6213..6230)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6213..6229)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6222..6244)
                     /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 ggatccgcca
      961 ccatgaacac tatccaacaa ttaatgatga ttttaaattc agcaagtgat caaccttcag
     1021 aaaatctgat ttcctatttt aacaactgca cagtgaatcc aaaagaaagt atactgaaaa
     1081 gagtgaagga tataggatac atctttaaag agaaatttgc taaagctgtg ggacagggtt
     1141 gtgtcgaaat tggatcacag cgatacaaac ttggagttcg cttgtattac cgagtaatgg
     1201 aatccatgct taaatcagaa gaagaacgat tatccattca aaattttagc aaacttctga
     1261 atgacaacat ttttcatatg tctttattgg cgtgcgctct tgaggttgta atggccacat
     1321 atagcagaag tacatctcag aatcttgatt ctggaacaga tttgtctttc ccatggattc
     1381 tgaatgtgct taatttaaaa gcctttgatt tttacaaagt gatcgaaagt tttatcaaag
     1441 cagaaggcaa cttgacaaga gaaatgataa aacatttaga acgatgtgaa catcgaatca
     1501 tggaatccct tgcatggctc tcagattcac ctttatttga tcttattaaa caatcaaagg
     1561 accgagaagg accaactgat caccttgaat ctgcttgtcc tcttaatctt cctctccaga
     1621 ataatcacac tgcagcagat atgtatcttt ctcctgtaag atctccaaag aaaaaaggtt
     1681 caactacgcg tgtaaattct actgcaaatg cagagacaca agcaacctca gccttccaga
     1741 cccagaagcc attgaaatct acctctcttt cactgtttta taaaaaagtg tatcggctag
     1801 cctatctccg gctaaataca ctttgtgaac gccttctgtc tgagcaccca gaattagaac
     1861 atatcatctg gacccttttc cagcacaccc tgcagaatga gtatgaactc atgagagaca
     1921 ggcatttgga ccaaattatg atgtgttcca tgtatggcat atgcaaagtg aagaatatag
     1981 accttaaatt caaaatcatt gtaacagcat acaaggatct tcctcatgct gttcaggaga
     2041 cattcaaacg tgttttgatc aaagaagagg agtatgattc tattatagta ttctataact
     2101 tggtcttcat gcagagactg aaaacaaata ttttgcagta tgcttccacc aggcccccta
     2161 ccttgtcacc aatacctcac attcctcgaa gcccttacaa gtttcctagt tcacccttac
     2221 ggattcctgg agggaacatc tatatttcac ccctgaagag tccatataaa atttcagaag
     2281 gtctgccaac accaacaaaa atgactccaa gatcaagaat cttagtgtca attggtgaat
     2341 cattcgggac ttctgagaag ttccagaaaa taaatcagat ggtatgtaac agcgaccgtg
     2401 tgctcaaaag aagtgctgaa ggaagcaacc ctcctaaacc actgaaaaaa ctacgctttg
     2461 atattgaagg atcagatgaa gcagatggaa gtaaacatct cccaggagag tccaaatttc
     2521 agcagaaact ggcagaaatg acttctactc gaacacgaat gcaaaagcag aaaatgaatg
     2581 atagcatgga tacctcaaac aaggaagaga aatgaggatc ggatccccgg gaattcatcg
     2641 tgactgactg acgatctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     2701 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     2761 tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag
     2821 cgatagcgga gtgtataatt cttgaagacg aaagggcctc gtgatacgcc tatttttata
     2881 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     2941 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     3001 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     3061 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     3121 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     3181 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     3241 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg
     3301 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     3361 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     3421 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     3481 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     3541 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc
     3601 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     3661 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     3721 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     3781 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     3841 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     3901 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     3961 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     4021 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     4081 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     4141 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     4201 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     4261 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     4321 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     4381 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     4441 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     4501 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     4561 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     4621 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     4681 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     4741 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     4801 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     4861 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc ataaattccg acaccatcga
     4921 atggtgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt
     4981 ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca
     5041 gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt
     5101 ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg
     5161 caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca
     5221 aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat
     5281 ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg
     5341 cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc
     5401 tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag
     5461 tattattttc tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg
     5521 tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct
     5581 ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg
     5641 cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt
     5701 tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac
     5761 cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga
     5821 cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca
     5881 aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct
     5941 gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc
     6001 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     6061 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
     6121 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
     6181 caggaaacag ctatgaccat gattacggat tcactggccg tcgttttaca acgtcgtgac
     6241 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     6301 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     6361 ggcgaatggc gctttgcctg gtttccggca ccagaagcgg tgccggaaag ctggctggag
     6421 tgcgatcttc ctgaggccga tactgtcgtc gtcccctcaa actggcagat gcacggttac
     6481 gatgcgccca tctacaccaa cgtaacctat cccattacgg tcaatccgcc gtttgttccc
     6541 acggagaatc cgacgggttg ttactcgctc acatttaatg ttgatgaaag ctggctacag
     6601 gaaggccaga cgcgaattat ttttgatggc gttggaatt
//
