LOCUS       sequence-258468-        6871 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 6871)
  AUTHORS   Krek W, Ewen ME, Shirodkar S, Arany Z, Kaelin WG, Livingston DM
  TITLE     Negative regulation of the growth-promoting transcription factor 
            E2F-1 by a stably bound cyclin A-dependent protein kinase.
  JOURNAL   Cell. 1994 Jul 15. 78(1):161-72.
  PUBMED    8033208
REFERENCE   2  (bases 1 to 6871)
  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. 1994 
            Jul 15. 78(1):161-72."
COMMENT     Sequence Label: HA-E2F-1 wt-pRcCMV
FEATURES             Location/Qualifiers
     source          1..6871
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             904..930
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             904..930
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             910..936
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     promoter        complement(2330..2348)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2331..2348)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2368..2385)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2374..2598
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2644..3157
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2731..2750)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2941..2962
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3180..3200)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3185..3514
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3365..3500
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3581..4375
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3635..3654)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4245..4264
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4549..4683
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4586..4605)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4640..4659
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4714..4730)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4714..4730)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4727..4749)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4738..4754
                     /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)."
     promoter        complement(join(4762..4768,4769..4786,4787..4792))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4807..4828
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4945..4962)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5116..5704)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5196..5215)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5875..6666,6667..6735))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6498..6517
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6736..6840)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttaccatg
      901 gcctacccct acgacgtgcc cgactacgcc tccctcggat cccatatggc cttggccggg
      961 gcccctgcgg gcggcccatg cgcgccggcg ctggaggccc tgctcggggc cggcgcgctg
     1021 cggctgctcg actcctcgca gatcgtcatc atctccgccg cgcaggacgc cagcgccccg
     1081 ccggctccca ccggccccgc ggcgcccgcc gccggcccct gcgaccctga cctgctgctc
     1141 ttcgccacac cgcaggcgcc ccggcccaca cccagtgcgc cgcggcccgc gctcggccgc
     1201 ccgccggtga agcggaggct ggacctggaa actgaccatc agtacctggc cgagagcagt
     1261 gggccagctc ggggcagagg ccgccatcca ggaaaaggtg tgaaatcccc gggggagaag
     1321 tcacgctatg agacctcact gaatctgacc accaagcgct tcctggagct gctgagccac
     1381 tcggctgacg gtgtcgtcga cctgaactgg gctgccgagg tgctgaaggt gcagaagcgg
     1441 cgcatctatg acatcaccaa cgtccttgag ggcatccagc tcattgccaa gaagtccaag
     1501 aaccacatcc agtggctggg cagccacacc acagtgggcg tcggcggacg gcttgagggg
     1561 ttgacccagg acctccgaca gctgcaggag agcgagcagc agctggacca cctgatgaat
     1621 atctgtacta cgcagctgcg cctgctctcc gaggacactg acagccagcg cctggcctac
     1681 gtgacgtgtc aggaccttcg tagcattgca gaccctgcag agcagatggt tatggtgatc
     1741 aaagcccctc ctgagaccca gctccaagcc gtggactctt cggagaactt tcagatctcc
     1801 cttaagagca aacaaggccc gatcgatgtt ttcctgtgcc ctgaggagac cgtaggtggg
     1861 atcagccctg ggaagacccc atcccaggag gtcacttctg aggaggagaa cagggccact
     1921 gactctgcca ccatagtgtc accaccacca tcatctcccc cctcatccct caccacagat
     1981 cccagccagt ctctactcag cctggagcaa gaaccgctgt tgtcccggat gggcagcctg
     2041 cgggctcccg tggacgagga ccgcctgtcc ccgctggtgg cggccgactc gctcctggag
     2101 catgtgcggg aggacttctc cggcctcctc cctgaggagt tcatcagcct ttccccaccc
     2161 cacgaggccc tcgactacca cttcggcctc gaggagggcg agggcatcag agacctcttc
     2221 gactgtgact ttggggacct cacccccctg gatttctgac aggatgcatg aattctgcag
     2281 atatccatca cactggcggc cgctcgagca tgcatctaga gggccctatt ctatagtgtc
     2341 acctaaatgc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     2401 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     2461 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     2521 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     2581 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctcg aggggggatc
     2641 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     2701 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     2761 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     2821 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     2881 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     2941 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     3001 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     3061 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta aatatttgct tatacaatct
     3121 tcctgttttt ggggcttttc tgattatcaa ccggggtggg taccgagctc gaattctgtg
     3181 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3241 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     3301 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     3361 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     3421 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     3481 aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt ggatatccat
     3541 tttcggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     3601 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     3661 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     3721 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct
     3781 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     3841 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     3901 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     3961 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     4021 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     4081 agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac
     4141 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     4201 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     4261 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     4321 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     4381 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     4441 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     4501 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     4561 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4621 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
     4681 atccgtcgac ctcgagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     4741 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     4801 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     4861 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     4921 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     4981 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     5041 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     5101 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     5161 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5221 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5281 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     5341 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5401 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5461 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5521 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     5581 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     5641 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5701 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     5761 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     5821 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     5881 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     5941 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     6001 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     6061 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     6121 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     6181 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     6241 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     6301 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6361 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6421 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6481 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6541 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6601 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     6661 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6721 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     6781 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6841 cacatttccc cgaaaagtgc cacctgacgt c
//
