LOCUS       sequence-10090-e        6508 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 6508)
  AUTHORS   Kaelin WG, Pallas DC, DeCaprio JA, Kaye FJ, Livingston DM
  TITLE     Identification of cellular proteins that can interact specifically 
            with the T/E1A-binding region of the retinoblastoma gene product.
  JOURNAL   Cell. 1991 Feb 8. 64(3):521-32.
  PUBMED    1825028
REFERENCE   2  (bases 1 to 6508)
  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. 1991 
            Feb 8. 64(3):521-32."
COMMENT     Sequence Label: Rb-379-928-Delta-703-737-pGex2T
FEATURES             Location/Qualifiers
     source          1..6508
                     /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(2558..2580)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2725..2743)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2811..2915
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2916..2984,2985..3776)
                     /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(3134..3153)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3947..4535
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4436..4455
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4689..4706
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4779..4856
                     /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             4857..5939
                     /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(4876..4895)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5952..5973
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5988..5993,5994..6011,6012..6018)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6026..6042
                     /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     6050..6066
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6050..6066
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(6062..6508,1..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(6082..6099)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6082..6098)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6091..6113)
                     /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 gatccgccac catgaacact atccaacaat
      961 taatgatgat tttaaattca gcaagtgatc aaccttcaga aaatctgatt tcctatttta
     1021 acaactgcac agtgaatcca aaagaaagta tactgaaaag agtgaaggat ataggataca
     1081 tctttaaaga gaaatttgct aaagctgtgg gacagggttg tgtcgaaatt ggatcacagc
     1141 gatacaaact tggagttcgc ttgtattacc gagtaatgga atccatgctt aaatcagaag
     1201 aagaacgatt atccattcaa aattttagca aacttctgaa tgacaacatt tttcatatgt
     1261 ctttattggc gtgcgctctt gaggttgtaa tggccacata tagcagaagt acatctcaga
     1321 atcttgattc tggaacagat ttgtctttcc catggattct gaatgtgctt aatttaaaag
     1381 cctttgattt ttacaaagtg atcgaaagtt ttatcaaagc agaaggcaac ttgacaagag
     1441 aaatgataaa acatttagaa cgatgtgaac atcgaatcat ggaatccctt gcatggctct
     1501 cagattcacc tttatttgat cttattaaac aatcaaagga ccgagaagga ccaactgatc
     1561 accttgaatc tgcttgtcct cttaatcttc ctctccagaa taatcacact gcagcagata
     1621 tgtatctttc tcctgtaaga tctccaaaga aaaaaggttc aactacgcgt gtaaattcta
     1681 ctgcaaatgc agagacacaa gcaacctcag ccttccagac ccagaagcca ttgaaatcta
     1741 cctctctttc actgttttat aaaaaagtgt atcggctagc ctatctccgg ctaaatacac
     1801 tttgtgaacg ccttctgtct gagcacccag aattagaaca tatcatctgg acccttttcc
     1861 agcacaccct gcagaatgag tatgaactca tgagagacag gcatttggac caaacattca
     1921 aacgtgtttt gatcaaagaa gaggagtatg attctattat agtattctat aacttggtct
     1981 tcatgcagag actgaaaaca aatattttgc agtatgcttc caccaggccc cctaccttgt
     2041 caccaatacc tcacattcct cgaagccctt acaagtttcc tagttcaccc ttacggattc
     2101 ctggagggaa catctatatt tcacccctga agagtccata taaaatttca gaaggtctgc
     2161 caacaccaac aaaaatgact ccaagatcaa gaatcttagt gtcaattggt gaatcattcg
     2221 ggacttctga gaagttccag aaaataaatc agatggtatg taacagcgac cgtgtgctca
     2281 aaagaagtgc tgaaggaagc aaccctccta aaccactgaa aaaactacgc tttgatattg
     2341 aaggatcaga tgaagcagat ggaagtaaac atctcccagg agagtccaaa tttcagcaga
     2401 aactggcaga aatgacttct actcgaacac gaatgcaaaa gcagaaaatg aatgatagca
     2461 tggatacctc aaacaaggaa gagaaatgag gatccccggg aattcatcgt gactgactga
     2521 cgatctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     2581 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     2641 cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag
     2701 tgtataattc ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     2761 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     2821 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     2881 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     2941 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     3001 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     3061 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     3121 cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac
     3181 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     3241 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     3301 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     3361 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     3421 aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc
     3481 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     3541 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     3601 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     3661 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     3721 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     3781 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     3841 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     3901 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     3961 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4021 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4081 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4141 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4201 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4261 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4321 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4381 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     4441 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     4501 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     4561 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     4621 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     4681 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     4741 ttacgcatct gtgcggtatt tcacaccgca taaattccga caccatcgaa tggtgcaaaa
     4801 cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga
     4861 aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc
     4921 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga
     4981 tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt
     5041 tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg
     5101 cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa
     5161 gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc
     5221 tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta
     5281 atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct
     5341 cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa
     5401 tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc
     5461 ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg
     5521 ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga
     5581 tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc
     5641 tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt
     5701 atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg
     5761 accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct
     5821 cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt
     5881 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     5941 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     6001 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     6061 tatgaccatg attacggatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     6121 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     6181 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg
     6241 ctttgcctgg tttccggcac cagaagcggt gccggaaagc tggctggagt gcgatcttcc
     6301 tgaggccgat actgtcgtcg tcccctcaaa ctggcagatg cacggttacg atgcgcccat
     6361 ctacaccaac gtaacctatc ccattacggt caatccgccg tttgttccca cggagaatcc
     6421 gacgggttgt tactcgctca catttaatgt tgatgaaagc tggctacagg aaggccagac
     6481 gcgaattatt tttgatggcg ttggaatt
//
