LOCUS       sequence-9988-ex        6504 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 6504)
  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 6504)
  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-738-775-pGex2T
FEATURES             Location/Qualifiers
     source          1..6504
                     /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(2554..2576)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2721..2739)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2807..2911
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2912..2980,2981..3772)
                     /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(3130..3149)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3943..4531
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4432..4451
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4685..4702
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4775..4852
                     /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             4853..5935
                     /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(4872..4891)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5948..5969
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5984..5989,5990..6007,6008..6014)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6022..6038
                     /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     6046..6062
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6046..6062
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(6058..6504,1..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(6078..6095)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6078..6094)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6087..6109)
                     /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 caaattatga
     1921 tgtgttccat gtatggcata tgcaaagtga agaatataga ccttaaattc aaaatcattg
     1981 taacagcata caaggatctt cctcatgctg ttcaggagcc ccctaccttg tcaccaatac
     2041 ctcacattcc tcgaagccct tacaagtttc ctagttcacc cttacggatt cctggaggga
     2101 acatctatat ttcacccctg aagagtccat ataaaatttc agaaggtctg ccaacaccaa
     2161 caaaaatgac tccaagatca agaatcttag tgtcaattgg tgaatcattc gggacttctg
     2221 agaagttcca gaaaataaat cagatggtat gtaacagcga ccgtgtgctc aaaagaagtg
     2281 ctgaaggaag caaccctcct aaaccactga aaaaactacg ctttgatatt gaaggatcag
     2341 atgaagcaga tggaagtaaa catctcccag gagagtccaa atttcagcag aaactggcag
     2401 aaatgacttc tactcgaaca cgaatgcaaa agcagaaaat gaatgatagc atggatacct
     2461 caaacaagga agagaaatga ggatcggatc cccgggaatt catcgtgact gactgacgat
     2521 ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     2581 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     2641 gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta
     2701 taattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     2761 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     2821 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     2881 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     2941 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     3001 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     3061 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     3121 taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg
     3181 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     3241 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     3301 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     3361 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     3421 cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa
     3481 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     3541 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     3601 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     3661 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     3721 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     3781 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     3841 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     3901 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     3961 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     4021 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     4081 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     4141 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     4201 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     4261 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     4321 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     4381 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     4441 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     4501 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     4561 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     4621 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     4681 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     4741 gcatctgtgc ggtatttcac accgcataaa ttccgacacc atcgaatggt gcaaaacctt
     4801 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc
     4861 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
     4921 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
     4981 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
     5041 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
     5101 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
     5161 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
     5221 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt
     5281 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca
     5341 tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
     5401 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa
     5461 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
     5521 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
     5581 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
     5641 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat
     5701 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
     5761 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact
     5821 ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     5881 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     5941 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     6001 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     6061 accatgatta cggattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     6121 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     6181 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcttt
     6241 gcctggtttc cggcaccaga agcggtgccg gaaagctggc tggagtgcga tcttcctgag
     6301 gccgatactg tcgtcgtccc ctcaaactgg cagatgcacg gttacgatgc gcccatctac
     6361 accaacgtaa cctatcccat tacggtcaat ccgccgtttg ttcccacgga gaatccgacg
     6421 ggttgttact cgctcacatt taatgttgat gaaagctggc tacaggaagg ccagacgcga
     6481 attatttttg atggcgttgg aatt
//
