LOCUS       sequence-197133-        6725 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian cell expression of the beta-galactosidase-specific 
            ubiquibody, R4-uAb.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6725)
  AUTHORS   Portnoff AD, Stephens EA, Varner JD, DeLisa MP
  TITLE     Ubiquibodies, synthetic E3 ubiquitin ligases endowed with unnatural 
            substrate specificity for targeted protein silencing.
  JOURNAL   J Biol Chem. 2014 Mar 14;289(11):7844-55. doi: 
            10.1074/jbc.M113.544825. Epub 2014 Jan 28.
  PUBMED    24474696
REFERENCE   2  (bases 1 to 6725)
  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"; doi: 
            "10.1074/jbc.M113.544825"; journalName: "J Biol Chem"; date: 
            "2014-03-14- 14"; volume: "289"; issue: "11"; pages: "7844-55"
COMMENT     Sequence Label: pcDNA3-R4-uAb
FEATURES             Location/Qualifiers
     source          1..6725
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1981..2004
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2008..2025
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        complement(2044..2062)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2045..2062)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2082..2099)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2088..2312
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2358..2786
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2445..2464)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2655..2676
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2795..2815)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2800..3129
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2980..3115
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3042..3061
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3196..3990
                     /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(3250..3269)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3860..3879
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4164..4285
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4201..4220)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4255..4274
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4334..4350)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4334..4350)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4347..4369)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4358..4374
                     /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(4382..4388,4389..4406,4407..4412))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4427..4448
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4565..4582)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4736..5324)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4816..4835)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5495..6286,6287..6355))
                     /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     6118..6137
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6356..6460)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6535..6554)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 accatggccg aggtgcagct ggtggagtct gggggaagcc tggtcaagcc tggggggtcc
      721 ctgagactct cctgtgcagc ctctggattc accttcagta actatagcat gaactgggtc
      781 cgccaggctc cagggaaggg gctggagtgg atctcatcca ttagtggtag tagtagatac
      841 atatactacg cagacttcgt gaagggccga ttcaccatct ccagagacaa cgccacgaac
      901 tcactgtacc tgcaaatgaa cagcctgaga gccgaggaca cggctgttta ttactgtgtg
      961 agatccagta ttacgatttt tggtggcggt atggacgtct ggggcagagg caccctggtc
     1021 accgtctcct caggtggagg cggttcaggc ggaggtggca gcggcggtgg cggatcgcag
     1081 tctgtgctga ctcagcctgc ctccgtgtct gggtctcctg gacagtcgat caccatctcc
     1141 tgcgctggaa ccagcagtga cgttggtggt tataactatg tctcctggta ccaacaacac
     1201 ccaggcaaag cccccaaact catgatttat gaggacagta agcggccctc aggggtttct
     1261 aatcgcttct ctggctccaa gtctggcaac acggcctccc tgacaatctc tgggctccag
     1321 gctgaggacg aggctgatta ttactgcagc tcatatacaa ccaggagcac tcgagttttc
     1381 ggcggaggga ccaagctggc cgtcctaggt gcggccgcag aattcggcag cggttctggc
     1441 cggctgaact tcggggacga catccccagc gctcttcgaa tcgcgaagaa gaagcgctgg
     1501 aacagcattg aggagcggcg catccaccag gagagcgagc tgcactccta cctctccagg
     1561 ctcattgccg cggagcgtga gagggagctg gaagagtgcc agcgaaacca cgagggtgat
     1621 gaggacgaca gccacgtccg ggcccagcag gcctgcattg aggccaagca cgacaagtac
     1681 atggcggaca tggacgagct tttttctcag gtggatgaga agaggaagaa gcgagacatc
     1741 cccgactacc tgtgtggcaa gatcagcttt gagctgatgc gggagccgtg catcacgccc
     1801 agtggcatca cctacgaccg caaggacatc gaggagcacc tgcagcgtgt gggtcatttt
     1861 gaccccgtga cccggagccc cctgacccag gaacagctca tccccaactt ggctatgaag
     1921 gaggttattg acgcattcat ctctgagaat ggctgggtgg aggattacgt cgacggagca
     1981 gactacaagg acgatgacga caagggacat catcatcatc atcactaatc tagagggccc
     2041 tattctatag tgtcacctaa atgctagagc tcgctgatca gcctcgactg tgccttctag
     2101 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     2161 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     2221 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     2281 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     2341 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2401 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     2461 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2521 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     2581 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     2641 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     2701 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     2761 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
     2821 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2881 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2941 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     3001 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     3061 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     3121 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca agagacagga
     3181 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     3241 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     3301 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     3361 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     3421 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     3481 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     3541 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     3601 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     3661 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     3721 gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     3781 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     3841 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     3901 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     3961 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     4021 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     4081 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     4141 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     4201 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4261 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     4321 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     4381 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     4441 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4501 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     4561 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     4621 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     4681 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     4741 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     4801 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     4861 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     4921 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     4981 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5041 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5101 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5161 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5221 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5281 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5341 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5401 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5461 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     5521 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5581 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     5641 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     5701 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     5761 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     5821 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     5881 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     5941 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6001 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6061 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6121 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6181 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6241 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6301 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6361 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6421 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     6481 cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat
     6541 ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc
     6601 tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat
     6661 gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac
     6721 gcgtt
//
