LOCUS       sequence-8365-ex        6771 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 6771)
  AUTHORS   Jin J, Ang XL, Shirogane T, Wade Harper J
  TITLE     Identification of substrates for F-box proteins.
  JOURNAL   Methods Enzymol. 2005;399:287-309. doi: 
            10.1016/S0076-6879(05)99020-4.
  PUBMED    16338364
REFERENCE   2  (bases 1 to 6771)
  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: "Methods 
            Enzymol."; date: "2005"; volume: "399:287-309. doi"; pages: " 
            10.1016/S0076-6879(05)99020-4"
COMMENT     Sequence Label: pcDNA3-DN-hCUL1-FLAG
FEATURES             Location/Qualifiers
     source          1..6771
                     /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             2272..2295
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2326..2344)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2327..2344)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2364..2381)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2370..2591
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2637..3065
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2724..2743)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2934..2955
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3074..3094)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3079..3409
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3260..3395
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3322..3341
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3476..4270
                     /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(3530..3549)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4140..4159
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4444..4565
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4481..4500)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4535..4554
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4614..4630)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4614..4630)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4627..4649)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4638..4654
                     /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(4662..4668,4669..4686,4687..4692))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4707..4728
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4845..4862)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5016..5604)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5096..5115)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5775..6566,6567..6635))
                     /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     6398..6417
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6636..6740)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct 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 atgggtggac 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 gcttggtacc
      901 gagctcggat ccaccatgtc gtcaacccgg agccagaacc cccacggcct gaagcagatt
      961 ggcctggacc agatctggga cgacctcaga gccggcatcc agcaggtgta cacacggcag
     1021 agcatggcca agtccagata tatggagctc tacactcatg tttataacta ctgtactagt
     1081 gttcaccagt caaaccaagc acgaggagct ggagttcctc cttctaagtc gaaaaagggg
     1141 cagacacctg gaggagctca gtttgttggc ctggaattat ataaacgact taaggaattt
     1201 ttgaagaatt acttgacaaa tcttcttaag gatggagaag atttgatgga tgagagtgta
     1261 ctgaaattct acactcaaca atgggaagat tatcgatttt caagcaaagt gctgaatgga
     1321 atttgtgcct acctcaatag acattgggtt cgccgtgaat gtgacgaagg acgaaaagga
     1381 atatatgaaa tctattcgct tgcattggtg acttggagag actgtctgtt caggccactg
     1441 aataaacagg taacaaatgc tgttttaaag ctgattgaaa aggaaaggaa tggtgaaacc
     1501 atcaatacaa gattgattag tggagttgta cagtcttacg tggaattggg gctgaatgaa
     1561 gatgatgcat ttgcaaaggg ccctacgtta acagtgtata aagaatcctt tgaatctcaa
     1621 tttttggctg acacagagag attttatacc agagagagta ctgaattctt gcagcagaac
     1681 ccagttactg aatatatgaa aaaggcagag gctcgtctgc ttgaggaaca acgaagagtt
     1741 caggtttacc ttcatgaaag cacacaagat gaattagcaa ggaaatgtga acaagtcctc
     1801 attgaaaaac acttggaaat tttccacaca gaatttcaga atttattgga tgctgacaaa
     1861 aatgaagatt tgggacgcat gtataatctt gtatctagaa tccaggatgg cctaggagaa
     1921 ttgaaaaaac tgttggagac acacattcat aatcagggtc ttgcagccat tgaaaagtgt
     1981 ggagaagctg ctttaaatga ccccaaaatg tatgtacaga cagtgcttga tgttcataaa
     2041 aaatacaatg ccctggtaat gtctgcattc aacaatgacg ctggctttgt ggctgctctt
     2101 gataaggctt gtggtcgctt cataaacaac aacgcggtta ccaagatggc ccaatcatcc
     2161 agtaaatccc ctgagttgct ggctcgatac tgtgactcct tgttgaagaa aagttccaag
     2221 aacccagagg aggcagaact agaagacaca ctcaatcaag tgatggttgt cgactacaag
     2281 gatgacgatg acaagtgact cgagcatgca tctagagggc cctattctat agtgtcacct
     2341 aaatgctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgtttg
     2401 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     2461 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     2521 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     2581 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     2641 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     2701 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     2761 cgccggcttt ccccgtcaag ctctaaatcg gggcatccct ttagggttcc gatttagtgc
     2821 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     2881 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     2941 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     3001 gattttgggg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     3061 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccaggc
     3121 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     3181 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     3241 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     3301 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct
     3361 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg
     3421 agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat
     3481 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     3541 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     3601 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga
     3661 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     3721 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     3781 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     3841 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     3901 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     3961 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga
     4021 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     4081 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     4141 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     4201 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     4261 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca
     4321 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     4381 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     4441 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     4501 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     4561 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag
     4621 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     4681 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     4741 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     4801 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     4861 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     4921 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     4981 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5041 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5101 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5161 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc
     5221 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5281 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5341 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5401 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
     5461 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5521 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5581 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5641 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5701 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     5761 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5821 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     5881 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     5941 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6001 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6061 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     6121 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6181 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6241 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6301 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6361 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     6421 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6481 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6541 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6601 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6661 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6721 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c
//
