LOCUS       Exported                8243 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    Flag-HA-USP2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8243)
  AUTHORS   Sowa ME, Bennett EJ, Gygi SP, Harper JW
  TITLE     Defining the human deubiquitinating enzyme interaction landscape.
  JOURNAL   Cell. 2009 Jul 23. 138(2):389-403.
  PUBMED    19615732
REFERENCE   2  (bases 1 to 8243)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8243
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             4..27
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             40..66
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    82..106
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1922..1946)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    1976..2528
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2120..2137)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2347..2366
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2540..3139
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2540..2559)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3036..3056
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3215..3729
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    3891..3907
                     /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(3915..3945)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3960..3981
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4098..4115)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4269..4857)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4349..4368)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5028..5888)
                     /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     5651..5670
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5889..5993)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6061..6079
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6117..6139)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6239..6258
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6452..6474
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6602..6621
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             6821..7337
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    7401..7742
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             7809..8225
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     8155..8177
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     8193..8209
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      8238..4
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
ORIGIN
        1 atggactaca aggatgacga tgacaagctc gatggaggat acccatacga tgttccagat
       61 tacgctgctc gaggttatca aacaagtttg tacaaaaaag ttggcatgtc ccagctctcc
      121 tccaccctga agcgctacac agaatcggcc cgctacacag atgcccacta tgccaagtcg
      181 ggctatggtg cctacacccc atcctcctat ggggccaatc tggctgcctc cttactggag
      241 aaggagaaac ttggtttcaa gccggtcccc accagcagct tcctcacccg tccccgtacc
      301 tatggcccct cctccctcct ggactatgac cggggccgcc ccctgctgag acccgacatc
      361 actgggggtg gtaagcgggc agagagccag acccggggta ctgagcggcc tttaggcagt
      421 ggcctcagcg ggggcagcgg attcccttat ggagtgacca acaactgcct cagctacctg
      481 cccatcaatg cctatgacca gggggtgacc ctaacccaga agctggacag ccaatcagac
      541 ctggcccggg atttctccag cctccggacc tcagatagct accggataga ccccaggaac
      601 ctgggccgca gccccatgct ggcccggacg cgcaaggagc tctgcaccct gcaggggctc
      661 taccagacag ccagctgccc tgaatacctg gtcgactacc tggagaacta tggtcgcaag
      721 ggcagtgcat ctcaggtgcc ctcccaggcc cctccctcac gagtccctga aatcatcagc
      781 ccaacctacc gacccattgg ccgctacacg ctgtgggaga cgggaaaggg tcaggcccct
      841 gggcccagcc gctccagctc cccgggaaga gacggcatga attctaagag tgcccagggt
      901 ctggctggtc ttcgaaacct tgggaacacg tgcttcatga actcaattct gcagtgcctg
      961 agcaacactc gggagttgag agattactgc ctccagaggc tctacatgcg ggacctgcac
     1021 cacggcagca atgcacacac agccctcgtg gaagagtttg caaaactaat tcagaccata
     1081 tggacttcat cccccaatga tgtggtgagc ccatctgagt tcaagaccca gatccagaga
     1141 tatgcaccgc gctttgttgg ctataatcag caggatgctc aggagttcct tcgctttctt
     1201 ctggatgggc tccataacga ggtgaaccga gtgacactga gacctaagtc caaccctgag
     1261 aacctcgatc atcttcctga tgacgagaaa ggccgacaga tgtggagaaa atatctagaa
     1321 cgggaagaca gtaggatcgg ggatctcttt gttgggcagc taaagagctc gctgacgtgt
     1381 acagattgtg gttactgttc tacggtcttc gaccccttct gggacctctc actgcccatt
     1441 gctaagcgag gttatcctga ggtgacatta atggactgca tgaggctctt caccaaagag
     1501 gatgtgcttg atggagatga aaagccaaca tgctgtcgct gccgaggcag aaaacggtgt
     1561 ataaagaagt tctccatcca gaggttccca aagatcttgg tgctccatct gaagcggttc
     1621 tcagaatcca ggatccgaac cagcaagctc acaacatttg tgaacttccc cctaagagac
     1681 ctggacttaa gagaatttgc ctcagaaaac accaaccatg ctgtttacaa cctgtacgct
     1741 gtgtccaatc actccggaac caccatgggt ggccactata cagcctactg tcgcagtcca
     1801 gggacaggag aatggcacac tttcaacgac tccagcgtca ctcccatgtc ctccagccaa
     1861 gtgcgcacca gcgacgccta cctgctcttc tacgaactgg ccagcccgcc ctcccgaatg
     1921 tacccaactt tcttgtacaa agtggttcga taacgaattc cgcccccccc ccctaacgtt
     1981 actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc
     2041 atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc
     2101 attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag
     2161 gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg
     2221 cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat
     2281 acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga
     2341 gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc
     2401 cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg
     2461 ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg aaaaacacga
     2521 tgataatatg gccacaacca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga
     2581 cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg
     2641 ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct
     2701 cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc
     2761 ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg
     2821 catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc
     2881 gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca
     2941 ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc
     3001 cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct
     3061 cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac
     3121 ccgcaagccc ggtgcctgat gcatcgatgt cgacctgcag ccaagcttat cgataaaata
     3181 aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg
     3241 caagctagct taagtaacgc cattttgcaa ggcatggaaa atacataact gagaatagag
     3301 aagttcagat caaggttagg aacagagaga cagcagaata tgggccaaac aggatatctg
     3361 tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggtccccag atgcggtccc
     3421 gccctcagca gtttctagag aaccatcaga tgtttccagg gtgccccaag gacctgaaat
     3481 gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt
     3541 ctgctccccg agctcaataa aagagcccac aacccctcac tcggcgcgcc agtcctccga
     3601 tagactgcgt cgcccgggta cccgtgtatc caataaaccc tcttgcagtt gcatccgact
     3661 tgtggtctcg ctgttccttg ggagggtctc ctctgagtga ttgactaccc gtcagcgggg
     3721 gtctttcatg ggtaacagtt tcttgaagtt ggagaacaac attctgaggg taggagtcga
     3781 atattaagta atcctgactc aattagccac tgttttgaat ccacatactc caatactcct
     3841 gaaatagttc attatggaca gcgcagaaag agctggggag aattgtgaaa ttgttatccg
     3901 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     3961 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4021 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4081 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     4141 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     4201 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     4261 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     4321 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     4381 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     4441 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     4501 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     4561 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4621 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     4681 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     4741 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4801 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4861 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4921 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     4981 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     5041 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     5101 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     5161 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     5221 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     5281 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     5341 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     5401 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     5461 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     5521 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     5581 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     5641 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     5701 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     5761 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     5821 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     5881 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     5941 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     6001 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     6061 aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc
     6121 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga
     6181 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg
     6241 gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc
     6301 gtaaggagaa aataccgcat caggcgccat tcgccattca ggctgcgcaa ctgttgggaa
     6361 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca
     6421 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcg
     6481 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     6541 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     6601 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     6661 cgatgcgtcc ggcgtagagg cgattagtcc aatttgttaa agacaggata tcagtggtcc
     6721 aggctctagt tttgactcaa caatatcacc agctgaagcc tatagagtac gagccataga
     6781 taaaataaaa gattttattt agtctccaga aaaagggggg aatgaaagac cccacctgta
     6841 ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc atggaaaata cataactgag
     6901 aatagagaag ttcagatcaa ggttaggaac agagagacag cagaatatgg gccaaacagg
     6961 atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg tccccagatg
     7021 cggtcccgcc ctcagcagtt tctagagaac catcagatgt ttccagggtg ccccaaggac
     7081 ctgaaatgac cctgtgcctt atttgaacta accaatcagt tcgcttctcg cttctgttcg
     7141 cgcgcttctg ctccccgagc tcaataaaag agcccacaac ccctcactcg gcgcgccagt
     7201 cctccgatag actgcgtcgc ccgggtaccc gtattcccaa taaagcctct tgctgtttgc
     7261 atccgaatcg tggactcgct gatccttggg agggtctcct cagattgatt gactgcccac
     7321 ctcgggggtc tttcatttgg aggttccacc gagatttgga gacccctgcc cagggaccac
     7381 cgaccccccc gccgggaggt aagctggcca gcggtcgttt cgtgtctgtc tctgtctttg
     7441 tgcgtgtttg tgccggcatc taatgtttgc gcctgcgtct gtactagtta gctaactagc
     7501 tctgtatctg gcggacccgt ggtggaactg acgagttctg aacacccggc cgcaaccctg
     7561 ggagacgtcc cagggacttt gggggccgtt tttgtggccc gacctgagga agggagtcga
     7621 tgtggaatcc gaccccgtca ggatatgtgg ttctggtagg agacgagaac ctaaaacagt
     7681 tcccgcctcc gtctgaattt ttgctttcgg tttggaaccg aagccgcgcg tcttgtctgc
     7741 tgcagcgctg cagcatcgtt ctgtgttgtc tctgtctgac tgtgtttctg tatttgtctg
     7801 aaaattaggg ccagactgtt accactccct taagtttgac cttaggtcac tggaaagatg
     7861 tcgagcggat cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct
     7921 gctctgcaga atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag
     7981 acctcatcac ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc
     8041 aggtccccta catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc
     8101 cctttgtaca ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg
     8161 aacctcctcg ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag
     8221 gcgccggaat tagatctgcc acc
//
