LOCUS       sequence-9989-ex        6187 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 6187)
  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 6187)
  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-409-815-pGex2T
FEATURES             Location/Qualifiers
     source          1..6187
                     /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"
     regulatory      936..945
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2237..2259)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2404..2422)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2490..2594
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2595..2663,2664..3455)
                     /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(2813..2832)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3626..4214
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4115..4134
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4368..4385
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4458..4535
                     /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             4536..5618
                     /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(4555..4574)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5631..5652
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5667..5672,5673..5690,5691..5697)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5705..5721
                     /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     5729..5745
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5729..5745
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5741..6187,1..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(5761..5778)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5761..5777)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5770..5792)
                     /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 catggtgaat ccaaaagaaa
      961 gtatactgaa aagagtgaag gatataggat acatctttaa agagaaattt gctaaagctg
     1021 tgggacaggg ttgtgtcgaa attggatcac agcgatacaa acttggagtt cgcttgtatt
     1081 accgagtaat ggaatccatg cttaaatcag aagaagaacg attatccatt caaaatttta
     1141 gcaaacttct gaatgacaac atttttcata tgtctttatt ggcgtgcgct cttgaggttg
     1201 taatggccac atatagcaga agtacatctc agaatcttga ttctggaaca gatttgtctt
     1261 tcccatggat tctgaatgtg cttaatttaa aagcctttga tttttacaaa gtgatcgaaa
     1321 gttttatcaa agcagaaggc aacttgacaa gagaaatgat aaaacattta gaacgatgtg
     1381 aacatcgaat catggaatcc cttgcatggc tctcagattc acctttattt gatcttatta
     1441 aacaatcaaa ggaccgagaa ggaccaactg atcaccttga atctgcttgt cctcttaatc
     1501 ttcctctcca gaataatcac actgcagcag atatgtatct ttctcctgta agatctccaa
     1561 agaaaaaagg ttcaactacg cgtgtaaatt ctactgcaaa tgcagagaca caagcaacct
     1621 cagccttcca gacccagaag ccattgaaat ctacctctct ttcactgttt tataaaaaag
     1681 tgtatcggct agcctatctc cggctaaata cactttgtga acgccttctg tctgagcacc
     1741 cagaattaga acatatcatc tggacccttt tccagcacac cctgcagaat gagtatgaac
     1801 tcatgagaga caggcatttg gaccaaatta tgatgtgttc catgtatggc atatgcaaag
     1861 tgaagaatat agaccttaaa ttcaaaatca ttgtaacagc atacaaggat cttcctcatg
     1921 ctgttcagga gacattcaaa cgtgttttga tcaaagaaga gaagtatgat tctattatag
     1981 tattctataa ctcggtcttc atgcagagac tgaaaacaaa tattttgcag tatgcttcca
     2041 ccaggccccc taccttgtca ccaatacctc acattcctcg aagcccttac aagtttccta
     2101 gttcaccctt acggattcct ggagggaaca tctatatttc acccctgaag agtccatata
     2161 aaatttgagg atccccggga attcatcgtg actgactgac gatctgcctc gcgcgtttcg
     2221 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     2281 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     2341 ggggcgcagc catgacccag tcacgtagcg atagcggagt gtataattct tgaagacgaa
     2401 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     2461 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     2521 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     2581 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     2641 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     2701 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     2761 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     2821 gcgcggtatt atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt
     2881 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     2941 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     3001 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     3061 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     3121 gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac
     3181 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     3241 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     3301 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     3361 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     3421 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     3481 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     3541 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     3601 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     3661 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     3721 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     3781 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     3841 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     3901 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     3961 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     4021 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     4081 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     4141 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     4201 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     4261 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     4321 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     4381 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     4441 cacaccgcat aaattccgac accatcgaat ggtgcaaaac ctttcgcggt atggcatgat
     4501 agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg
     4561 tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc
     4621 acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc
     4681 ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct
     4741 ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc
     4801 aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag
     4861 cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg
     4921 atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg
     4981 atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac
     5041 tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat
     5101 taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc
     5161 aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa
     5221 ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga
     5281 tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct
     5341 cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca
     5401 tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc
     5461 agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca
     5521 ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     5581 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
     5641 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
     5701 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacggattc
     5761 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     5821 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     5881 cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc tttgcctggt ttccggcacc
     5941 agaagcggtg ccggaaagct ggctggagtg cgatcttcct gaggccgata ctgtcgtcgt
     6001 cccctcaaac tggcagatgc acggttacga tgcgcccatc tacaccaacg taacctatcc
     6061 cattacggtc aatccgccgt ttgttcccac ggagaatccg acgggttgtt actcgctcac
     6121 atttaatgtt gatgaaagct ggctacagga aggccagacg cgaattattt ttgatggcgt
     6181 tggaatt
//
