LOCUS       sequence-228216-        7198 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 7198)
  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 7198)
  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-hCUL4B-FLAG
FEATURES             Location/Qualifiers
     source          1..7198
                     /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             2698..2721
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2751..2769)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2752..2769)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2789..2806)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2795..3019
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3065..3493
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3152..3171)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3362..3383
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3502..3522)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3507..3836
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3687..3822
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3749..3768
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3903..4697
                     /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(3957..3976)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4567..4586
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4871..4992
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4908..4927)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4962..4981
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5041..5057)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5041..5057)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5054..5076)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5065..5081
                     /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(5089..5095,5096..5113,5114..5119))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5134..5155
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5272..5289)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5443..6031)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5523..5542)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6202..6993,6994..7062))
                     /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     6825..6844
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7063..7167)
                     /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 gcttggtacc
      901 gagctcggat ccaccatgat gtcacagtca tctggatcag gagatgggaa tgatgatgag
      961 gctactacct ctaaagacgg tggtttttct tcccccagtc cctcagctgc tgctgctgct
     1021 caggaggtca gatctgccac tgatggtaat accagcacca ctccgcccac ctctgccaag
     1081 aagagaaagt taaacagcag cagcagtagc agcagtaaca gtagtaacga gagagaagac
     1141 tttgattcca cctcttcctc ctcttccact cctcctttac aacccaggga ttcggcatcc
     1201 ccttcaacct cgtccttctg cctgggggtt tcagtggctg cttccagcca cgtaccgata
     1261 cagaagaagc tgcgttttga agacaccctg gagtttgtag ggtttgatgc gaagatggct
     1321 gaggaatcct cctcctcctc ctcctcatct tcaccaactg ctgcaacatc tcagcagcag
     1381 caacttaaaa ataagagtat attaatctct tctgtggctt cggtgcatca tgcaaacggc
     1441 ctagccaaat cttctaccac cgtctctagc tttgctaaca gcaaacctgg ctctgctaag
     1501 aagttagtga tcaagaactt taaagataag cctaaattac cagaaaacta cacagatgaa
     1561 acctggcaaa aactgaaaga agcagtggaa gctattcaga atagtacttc aattaagtac
     1621 aatttagaag aactctacca ggctgtagaa aatctctgtt cttacaagat ttctgcaaac
     1681 ttgtacaaac agctgagaca gatctgcgaa gatcacatca aagcacagat tcatcaattc
     1741 agagaggatt cattggatag cgttcttttt ttaaagaaga ttgatagatg ctggcaaaac
     1801 cattgcagac aaatgatcat gatcaggagc atttttttgt ttctggatag aacttacgtt
     1861 cttcagaatt caatgctacc ctccatttgg gacatgggac tggagttatt tagggctcat
     1921 attataagtg atcagaaagt gcagaataag acaattgatg gcattcttct cttgattgag
     1981 agggaaagga atggtgaagc aattgataga agtttacttc gaagcctttt aagcatgctg
     2041 tctgatttgc aaatttatca agattctttt gaacaacgat ttttggaaga aactaaccgg
     2101 ctctatgcag ctgaaggcca aaaattaatg caagaaagag aggttcctga atatctacat
     2161 catgttaaca aacgtctaga agaagaagca gacagactta ttacttactt agatcagacc
     2221 acccagaagt cattaattgc tactgtagaa aaacaacttc taggtgaaca cttaacagca
     2281 attcttcaga aaggtttaaa taacctcctt gatgaaaacc gaattcaaga tttgtctctt
     2341 ctgtatcagc tcttcagtag agttcgaggt ggagttcagg ttcttttgca gcagtggatc
     2401 gaatatatca aggcatttgg cagcactatt gtaattaatc ctgaaaaaga taaaaccatg
     2461 gttcaagaat tgctggattt taaagataag gttgaccata taattgatat ctgctttctg
     2521 aagaatgaga aatttatcaa tgccatgaaa gaagcatttg aaacgttcat taacaaaaga
     2581 ccaaataaac cagctgaact tatagctaag tatgtagatt caaaacttcg tgcaggcaac
     2641 aaagaagcta cagatgaaga acttgagaaa atgttggata aaattatgat catatttgac
     2701 tacaaggatg acgatgacaa gtagtcgagc atgcatctag agggccctat tctatagtgt
     2761 cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     2821 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     2881 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     2941 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     3001 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat
     3061 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     3121 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     3181 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     3241 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     3301 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     3361 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     3421 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     3481 tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     3541 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     3601 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     3661 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     3721 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct
     3781 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     3841 tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc
     3901 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     3961 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     4021 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     4081 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     4141 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     4201 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     4261 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     4321 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     4381 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg
     4441 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     4501 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     4561 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     4621 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     4681 ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa
     4741 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     4801 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     4861 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     4921 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     4981 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     5041 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     5101 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     5161 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     5221 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     5281 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     5341 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     5401 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5461 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5521 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5581 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     5641 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5701 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5761 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5821 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5881 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5941 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6001 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     6061 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     6121 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     6181 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6241 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6301 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6361 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6421 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6481 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6541 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6601 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6661 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6721 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6781 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6841 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6901 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6961 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7021 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     7081 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     7141 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
