LOCUS       sequence-132794-        9605 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 9605)
  AUTHORS   Magnifico A, Ettenberg S, Yang C, Mariano J, Tiwari S, Fang S, 
            Lipkowitz S, Weissman AM
  TITLE     WW domain HECT E3s target Cbl RING finger E3s for proteasomal 
            degradation.
  JOURNAL   J Biol Chem. 2003 Oct 31. 278(44):43169-77.
  PUBMED    12907674
REFERENCE   2  (bases 1 to 9605)
  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: "J Biol 
            Chem. 2003 Oct 31. 278(44):43169-77."
COMMENT     Sequence Label: pCINeo-myc-Itch/2859
FEATURES             Location/Qualifiers
     source          1..9605
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(30..52)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     152..171
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     polyA_signal    212..260
                     /note="synthetic polyadenylation signal"
     CDS             complement(324..1118)
                     /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(435..454)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1045..1064
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(1169..1526)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1183..1318
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        1335..1526
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     1483..1503
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(1643..2098)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1780..1801)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1992..2011
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    2284..2405
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2321..2340)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2375..2394
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(6559..6588)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     regulatory      6588..6597
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6629..6647)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6690..6709)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          6692..6824
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     complement(6964..6988)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(6985..7196)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(7014..7034)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        7197..7576
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(7787..8375)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7867..7886)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8546..9337,9338..9406))
                     /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     9169..9188
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9407..9511)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     9579..9597
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
       61 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
      121 gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg
      181 caccatacgc ggatccttat cgctatcgat tcacacaaaa aaccaacaca cagatgtaat
      241 gaaaataaag atattttatt gcggccatcg tgatggcagg ttgggcgtcg cttggtcggt
      301 catttcgaac cccagagtcc cgctcagaag aactcgtcaa gaaggcgata gaaggcgatg
      361 cgctgcgaat cgggagcggc gataccgtaa agcacgagga agcggtcagc ccattcgccg
      421 ccaagctctt cagcaatatc acgggtagcc aacgctatgt cctgatagcg gtccgccaca
      481 cccagccggc cacagtcgat gaatccagaa aagcggccat tttccaccat gatattcggc
      541 aagcaggcat cgccatgggt cacgacgaga tcctcgccgt cgggcatgcg cgccttgagc
      601 ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg
      661 acaagaccgg cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg
      721 aatgggcagg tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat
      781 actttctcgg caggagcaag gtgagatgac aggagatcct gccccggcac ttcgcccaat
      841 agcagccagt cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaacgccc
      901 gtcgtggcca gccacgatag ccgcgctgcc tcgtcctgca gttcattcag ggcaccggac
      961 aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca
     1021 tcagagcagc cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg
     1081 gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgg tggctctagc cttaagttcg
     1141 agactgttgt gtcagaagaa tcaagctttt tgcaaaagcc taggcctcca aaaaagcctc
     1201 ctcactactt ctggaatagc tcagaggccg aggcggcctc ggcctctgca taaataaaaa
     1261 aaattagtca gccatggggc ggagaatggg cggaactggg cggagttagg ggcgggatgg
     1321 gcggagttag gggcgggact atggttgctg actaattgag atgcatgctt tgcatacttc
     1381 tgcctgctgg ggagcctggg gactttccac acctggttgc tgactaattg agatgcatgc
     1441 tttgcatact tctgcctgct ggggagcctg gggactttcc acaccctaac tgacacacat
     1501 tccacagctg gttctttccg cctcagaagg tacctaacca agttcctctt tcagaggtta
     1561 tttcaggcca tggtgctgcg cagatccgcg tatgcggtgt gaaataccgc acagatgcgt
     1621 aaggagaaaa taccgcatca ggaaattgta agcgttaata ttttgttaaa attcgcgtta
     1681 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     1741 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     1801 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     1861 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     1921 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     1981 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     2041 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc
     2101 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     2161 tacgccagcc cggatcgatc cttatcggat tttaccacat ttgtagaggt tttacttgct
     2221 ttaaaaaacc tcccacatct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     2281 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     2341 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     2401 tcttatcatg tctgctcgaa gcattaaccc tcactaaagg gaagcggccg ctctagagat
     2461 gacatgtatt tcttgtggta taagggtaaa aagagggctg gggaggagcc gagctctccc
     2521 ttcactgatc actttttcat gaggcaaccc gtctgtcacg tctctgctga ggatggcagt
     2581 gttaaggagc acagctgcac gagttcaaac tgtcccagga tgagaacagg acgagcagct
     2641 agggacacag aaacactttc tttcctatgg gacaggggtt tgtcacactc ggtgttcttg
     2701 gctttatgct atgccaccaa cacatttcaa ggtccccatt agagaaataa ggctaatttt
     2761 gggatttggc tacaataggg actcacctgg ctgaggtttc atcttgctgc ccataaagtc
     2821 tgtggcagca accatcctca cagtctaatg ctaagccaag gtaaatgacc tacagagaag
     2881 gtcaaagtgc attcatcagt tactaaagga gcagcctaag ataaaggagt catggaaaga
     2941 aaagggaaat ctacaatttt taggggagag aaaaatattg tagatcagga ccatgtaaga
     3001 gggtagacac ggctgctcct gtgtcaggag tggagtgtga agacagcgca gctgtgtgct
     3061 gctggacgct gttgtgactg cagagtctgc accactgacc tccatttgtg ttaggaaatg
     3121 gttgcaactt gaatagttta gcaaaaatga aatgtaacta aggtgttgga ttacttcaac
     3181 accataaagc ttcaatttat gtgtaggatt taaaaaaatc aagaatgatc ttagatgaat
     3241 tcatctgaac ttattttaat tctaggttaa tttctaaaaa cgtgaaggaa ccacagtgct
     3301 gatactgttt gtaaagttta gtatttgcaa ggcaatagaa caattacaaa ctgaatgaag
     3361 agaggtatgg tcatatgttg gcggggacca agttttccct gaattcaaat agacaaaact
     3421 actttgtaga aaaatgcaag tcctgcatca cttccaggaa tagcatcaga atggtgcact
     3481 gattcatgct ccaggaatga aaaacaacag tcaggccagg ctgggacgag cggagagcac
     3541 aagcagcagc tgctcatgtg cccagacact gagttaagct tgcacctctg agcgaggctt
     3601 tacaggaaga ccttgtgtat tgcgtttccg agatgtccct gcagaatcct ttacaacacc
     3661 cactaagaga cctatgccca agtgagactt cggacataca gcaaactgtt gttaagggaa
     3721 caatctagtg gaataagacg agggaaattt cctcgctgtg ttaaatctca tgttaagttt
     3781 ctgactacag tttgtagatc tgctttctgg aaacgttgag tagaaatcca gaatgactgg
     3841 aagatctcaa cgataacatt aatttactta ctctcaggta actctctagt cacagtccaa
     3901 gtttacaata agatgtgcaa caggcagaga aggaagggga cagacactgc agacaagtcc
     3961 ctggcctctg gtggcttaag gtctaaacaa ttactcttgt ccaaatcctt cagtttcttc
     4021 aatggcaaat aacagctttt ccttcagttg ctcatagctc ttgtaaggtg ggaggtccag
     4081 gcggttaaaa caagtatggc ttctgggtaa ccaattttct ttgccaactt tttcgatgca
     4141 gaacttctgt ggtccattac tccccataag gtcagcaaat cctcccactg gcaatcggca
     4201 ggttccagta acaaactgca gaagtctcat cctcttctca ttatcaattt ctttaacaaa
     4261 ctgccaaaac cacatgatct gcttgcttgt tctggtgtag tggcggtaga tggcatgtct
     4321 ctgccagtca ttcaaatcaa tctcctgcat cccacacaga agaacctcta attccttcgc
     4381 atcaaaatat tgcaaatatt gctgaggaag aatttcatta aagccctcaa agaaagcttg
     4441 tgtctgttct tcaacacctc gagacaacct ccactcagct accatcctga tgtattcctc
     4501 cttgttttct tctgtcacaa gaatattgcc accattcggt ttcaaatcat gactcttaat
     4561 ttcacctaga atttctttat caactgagaa gtacatttcc aaaccacatt cttcgatgtt
     4621 gttttcttta acccagatga gcgaattata aaattctgga tcaatagatt ctaaatcctt
     4681 aagtccaact ggtttattca ggatacgttt atagaatggt aaagaaaatc cggtatctat
     4741 gaatttccca tgaaacagag ccatggcaat aaatctgcca ataaaacgaa agtatttcag
     4801 gtggtctgga ttgatgtaag aagcggggtt tatctgcagg cagtagttat ccttccctgc
     4861 atattcaaac aggcaataca ttgggttcaa cacttcatgt gacaaaagaa agaaccattc
     4921 tcttgctaca cctccataat ctaaaccttc ttctcctggg aaaatcaccc acaaacgtct
     4981 tctcaggtct tgcgggctga agctcatgat ctgctgaaag gaatcctcaa acaatgtttt
     5041 tcttgtcact gtgatcttta tatgctgtgg catggccagt tgctggcacc agaaccggaa
     5101 atactgaact tttgccttga agtcccgcac ataggctatc tggggcccat tgtctaaggc
     5161 tgattttccc gttcgtggat ctatataagt agttgctctt ctattgtggt ccacaaaata
     5221 tggaattcca tccactgtga atctcatttc ccagccttct ggtaagggct tttcatttaa
     5281 ctgaccttgg cttctggggt cttcccactg agtaatccga gtgttgtggt tgacaaaata
     5341 cactctgcca ttgctatcag ttctcttctc ccatccaggg ggcaacgggc caagcggatc
     5401 aaattctttg ttttgtgatg tagcaaacaa atcttgattc ccatagatga atctctggtt
     5461 aaactgctgc attgctccct gaagctgact acgctgtagc tgccactgtt catagttccg
     5521 gacagattcc aaggttggcc tctgccatgt tgtggttctt gtgaaatgat caacataata
     5581 aatacgtccc atattgtcca cacgccgttc ccagccagga ggtagaggtt ctggtctatc
     5641 ccatgttgtt cgcttttcaa catggtctac atagtaaaca cgcccatgct ggtctactct
     5701 ctgctcccac ccaggtggta ggggagcttg gcttacagta ttcagaggcc tagggcccga
     5761 gcctccagat atagtaagag gaattattaa tccagatgtt gctccttcag atgtacttgt
     5821 atttacattt gtatttgttg gtggcaagga gcctgtacta gatccatcac tgtcagaatt
     5881 cgtggatggt gagccattga cagaagctgg tcttcgtggg gtgggtggag gtggtctcga
     5941 aggtctagga ggtctagaag gcttaaaacc accatttgaa agggacggag aattgttccc
     6001 attggccctc ttgttttccc ctgacgctgc aacctcaggg tcctctgatc catttgtgct
     6061 cactcttgta tcatctctgg ttctgcagcc atcatcattc tgtgtagtac tctcggagca
     6121 tgacgtttca ccgttagtaa caacctcagc ttctacttgc agcccatcaa gacaaactga
     6181 caaatctccc atcgtctctg ttggctcttt gtcacctaca agctgcaaag tcataactac
     6241 ttcttcaagt ttcatattgt ttgactttaa tgtttcataa atatctaatc cagcagttcc
     6301 caataaaaca tcagacttca gggtctggtg actccacaca cgaaaacata atttactcgt
     6361 aggggtaaca ataactgtga ggggctgctt ccacttggga ctgtttgtat tattgcattt
     6421 ttctgtcttc tttgactgtc catctactgt gacttctaca taaggacttg gtccaaacca
     6481 attcttttta ttttctttaa gtttggctga gatgacagtg atctgaagtt gagatttcat
     6541 ggtcagacta ccaccgttca ggtcctcctc ggaaatcagc ttctgctcca tggtggcggc
     6601 tggtaccaag cttaagttta aacgctagcc tatagtgagt cgtattaagt actctagcct
     6661 taagagctgt aattgaactg ggagtggaca cctgtggaga gaaaggcaaa gtggatgtca
     6721 gtaagaccaa taggtgccta tcagaaacgc aagagtcttc tctgtctcga caagcccagt
     6781 ttctattggt ctccttaaac ctgtcttgta accttgatac ttacctgccc agtgcctcac
     6841 gaccaacttc tgcaaggcta ccttaagaga gtcactgcag cttaagttcg agactgttgt
     6901 gtcagaagca ctgactgcgt tagcaattta actgtgataa actaccgcaa taaagcttct
     6961 agtgatctga cggttcacta aacgagctct gcttatatag acctcccacc gtacacgcct
     7021 accgcccatt tgcgtcaacg gggcgggcga tcgcagttgt tacgacattt tggaaagtcc
     7081 cgttgatttt ggtgccaaaa caaactccca ttgacgtcaa tggggtggag acttggaaat
     7141 ccccgtgagt caaaccgcta tccacgccca ttggtgtact gccaaaaccg catcaccatg
     7201 gtaatagcga tgactaatac gtagatgtac tgccaagtag gaaagtcccg taaggtcatg
     7261 tactgggcat aatgccaggc gggccattta ccgtcattga cgtcaatagg gggcggactt
     7321 ggcatatgat acacttgatg tactgccaag tgggcagttt accgtaaata ctccacccat
     7381 tgacgtcaat ggaaagtccc tattggcgtt actatgggaa catacgtcat tattgacgtc
     7441 aatgggcggg ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg
     7501 cggaactcca tatatgggct atgaactaat gaccccgtaa ttgattacta ttaataacta
     7561 gtcaataatc aatgccaaca tggcggtcat attggacatg agccaatata aatgtacata
     7621 ttatgatata gatacaacgt atgcaatggc caatagccaa tattgattta tgctatataa
     7681 ccaatgaata atatggctaa tggccaatat tgaagatctg tcgagccatg tgagcaaaag
     7741 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     7801 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     7861 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     7921 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     7981 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     8041 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     8101 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     8161 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     8221 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     8281 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     8341 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     8401 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     8461 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     8521 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     8581 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     8641 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     8701 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     8761 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     8821 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     8881 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     8941 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     9001 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     9061 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     9121 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     9181 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     9241 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     9301 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     9361 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     9421 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     9481 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     9541 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
     9601 ttcgt
//
