LOCUS       sequence-9981-ex        6226 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 6226)
  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 6226)
  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-379-792-pGex2TK
FEATURES             Location/Qualifiers
     source          1..6226
                     /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(2276..2298)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2443..2461)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2529..2633
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2634..2702,2703..3494)
                     /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(2852..2871)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3665..4253
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4154..4173
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4407..4424
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4497..4574
                     /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             4575..5657
                     /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(4594..4613)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5670..5691
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5706..5711,5712..5729,5730..5736)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5744..5760
                     /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     5768..5784
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5768..5784
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5780..6226,1..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(5800..5817)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5800..5816)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5809..5831)
                     /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 cggtcttcat gcagagactg aaaacaaata ttttgcagta tgcttccacc aggcccccta
     2161 ccttgtcacc aatacctcac attcctcgaa gcccttacaa gttttgagga tccccgggaa
     2221 ttcatcgtga ctgactgacg atctgcctcg cgcgtttcgg tgatgacggt gaaaacctct
     2281 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     2341 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt
     2401 cacgtagcga tagcggagtg tataattctt gaagacgaaa gggcctcgtg atacgcctat
     2461 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     2521 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     2581 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     2641 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     2701 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     2761 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     2821 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg
     2881 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     2941 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     3001 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     3061 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     3121 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag
     3181 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     3241 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     3301 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     3361 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     3421 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     3481 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     3541 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     3601 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     3661 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     3721 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     3781 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     3841 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     3901 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     3961 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     4021 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     4081 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     4141 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     4201 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     4261 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     4321 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     4381 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     4441 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata aattccgaca
     4501 ccatcgaatg gtgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat
     4561 tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct
     4621 cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg
     4681 aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac
     4741 tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc
     4801 cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg
     4861 tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg
     4921 cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg
     4981 tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca
     5041 tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg
     5101 cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc
     5161 tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac
     5221 gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg
     5281 gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg
     5341 ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata
     5401 ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc
     5461 tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca
     5521 atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa
     5581 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     5641 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
     5701 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
     5761 tttcacacag gaaacagcta tgaccatgat tacggattca ctggccgtcg ttttacaacg
     5821 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     5881 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     5941 cctgaatggc gaatggcgct ttgcctggtt tccggcacca gaagcggtgc cggaaagctg
     6001 gctggagtgc gatcttcctg aggccgatac tgtcgtcgtc ccctcaaact ggcagatgca
     6061 cggttacgat gcgcccatct acaccaacgt aacctatccc attacggtca atccgccgtt
     6121 tgttcccacg gagaatccga cgggttgtta ctcgctcaca tttaatgttg atgaaagctg
     6181 gctacaggaa ggccagacgc gaattatttt tgatggcgtt ggaatt
//
