LOCUS       sequence-223080-        7145 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 7145)
  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 7145)
  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: "Cell. 2009 
            Jul 23. 138(2):389-403."
COMMENT     Sequence Label: Flag-HA-GFP
FEATURES             Location/Qualifiers
     source          1..7145
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             173..689
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    753..1094
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             1161..1577
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1507..1529
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1545..1561
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      1590..1599
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1599..1622
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1635..1661
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    1677..1701
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(1701..1703,1704..1706,1707..2420)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1701..1703,1704..1706,1707..2417)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1746..1767)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2007..2026)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2354..2375
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2419..2443)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    2456..3023
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    2473..3025
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2617..2634)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2844..2863
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3037..3636
                     /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(3037..3056)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3533..3553
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3712..4226
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4388..4404
                     /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(4412..4418,4419..4436,4437..4442))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4457..4478
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4595..4612)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4766..5354)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4846..4865)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5525..6316,6317..6385))
                     /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     6148..6167
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6386..6490)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6558..6576
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6614..6636)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6736..6755
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6949..6971
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7099..7118
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tgatgccggc cacgatgcgt ccggcgtaga ggcgattagt ccaatttgtt aaagacagga
       61 tatcagtggt ccaggctcta gttttgactc aacaatatca ccagctgaag cctatagagt
      121 acgagccata gataaaataa aagattttat ttagtctcca gaaaaagggg ggaatgaaag
      181 accccacctg taggtttggc aagctagctt aagtaacgcc attttgcaag gcatggaaaa
      241 tacataactg agaatagaga agttcagatc aaggttagga acagagagac agcagaatat
      301 gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc aagaacagat
      361 ggtccccaga tgcggtcccg ccctcagcag tttctagaga accatcagat gtttccaggg
      421 tgccccaagg acctgaaatg accctgtgcc ttatttgaac taaccaatca gttcgcttct
      481 cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa agagcccaca acccctcact
      541 cggcgcgcca gtcctccgat agactgcgtc gcccgggtac ccgtattccc aataaagcct
      601 cttgctgttt gcatccgaat cgtggactcg ctgatccttg ggagggtctc ctcagattga
      661 ttgactgccc acctcggggg tctttcattt ggaggttcca ccgagatttg gagacccctg
      721 cccagggacc accgaccccc ccgccgggag gtaagctggc cagcggtcgt ttcgtgtctg
      781 tctctgtctt tgtgcgtgtt tgtgccggca tctaatgttt gcgcctgcgt ctgtactagt
      841 tagctaacta gctctgtatc tggcggaccc gtggtggaac tgacgagttc tgaacacccg
      901 gccgcaaccc tgggagacgt cccagggact ttgggggccg tttttgtggc ccgacctgag
      961 gaagggagtc gatgtggaat ccgaccccgt caggatatgt ggttctggta ggagacgaga
     1021 acctaaaaca gttcccgcct ccgtctgaat ttttgctttc ggtttggaac cgaagccgcg
     1081 cgtcttgtct gctgcagcgc tgcagcatcg ttctgtgttg tctctgtctg actgtgtttc
     1141 tgtatttgtc tgaaaattag ggccagactg ttaccactcc cttaagtttg accttaggtc
     1201 actggaaaga tgtcgagcgg atcgctcaca accagtcggt agatgtcaag aagagacgtt
     1261 gggttacctt ctgctctgca gaatggccaa cctttaacgt cggatggccg cgagacggca
     1321 cctttaaccg agacctcatc acccaggtta agatcaaggt cttttcacct ggcccgcatg
     1381 gacacccaga ccaggtcccc tacatcgtga cctgggaagc cttggctttt gacccccctc
     1441 cctgggtcaa gccctttgta caccctaagc ctccgcctcc tcttcctcca tccgccccgt
     1501 ctctccccct tgaacctcct cgttcgaccc cgcctcgatc ctccctttat ccagccctca
     1561 ctccttctct aggcgccgga attagatctg ccaccatgga ctacaaggat gacgatgaca
     1621 agctcgatgg aggataccca tacgatgttc cagattacgc tgctcgaggt tatcaaacaa
     1681 gtttgtacaa aaaagttggc atggtgagca agggcgagga gctgttcacc ggggtggtgc
     1741 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
     1801 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     1861 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
     1921 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     1981 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     2041 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     2101 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     2161 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     2221 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     2281 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
     2341 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     2401 tggacgagct gtacaagtga ccaactttct tgtacaaagt ggttcgataa cgaattccgc
     2461 cccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct
     2521 atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc
     2581 ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc
     2641 tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg
     2701 tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa
     2761 agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt
     2821 ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg
     2881 atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtgc acatgcttta
     2941 catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt
     3001 tcctttgaaa aacacgatga taatatggcc acaaccatga ccgagtacaa gcccacggtg
     3061 cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc
     3121 gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag
     3181 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac
     3241 gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc
     3301 gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag
     3361 atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc
     3421 ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg
     3481 gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc
     3541 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg
     3601 cgcacctggt gcatgacccg caagcccggt gcctgatgca tcgatgtcga cctgcagcca
     3661 agcttatcga taaaataaaa gattttattt agtctccaga aaaagggggg aatgaaagac
     3721 cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc atggaaaata
     3781 cataactgag aatagagaag ttcagatcaa ggttaggaac agagagacag cagaatatgg
     3841 gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg
     3901 tccccagatg cggtcccgcc ctcagcagtt tctagagaac catcagatgt ttccagggtg
     3961 ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt tcgcttctcg
     4021 cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac ccctcactcg
     4081 gcgcgccagt cctccgatag actgcgtcgc ccgggtaccc gtgtatccaa taaaccctct
     4141 tgcagttgca tccgacttgt ggtctcgctg ttccttggga gggtctcctc tgagtgattg
     4201 actacccgtc agcgggggtc tttcatgggt aacagtttct tgaagttgga gaacaacatt
     4261 ctgagggtag gagtcgaata ttaagtaatc ctgactcaat tagccactgt tttgaatcca
     4321 catactccaa tactcctgaa atagttcatt atggacagcg cagaaagagc tggggagaat
     4381 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     4441 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     4501 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4561 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4621 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4681 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4741 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4801 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4861 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4921 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4981 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     5041 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     5101 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5161 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     5221 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     5281 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     5341 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     5401 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     5461 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     5521 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5581 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5641 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     5701 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5761 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5821 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5881 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     5941 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     6001 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     6061 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     6121 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     6181 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     6241 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6301 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6361 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6421 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6481 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     6541 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg
     6601 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     6661 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     6721 gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg
     6781 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgccattcg ccattcaggc
     6841 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
     6901 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
     6961 gttgtaaaac gacggcgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
     7021 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
     7081 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
     7141 gccgg
//
