LOCUS       sequence-59658-e        6253 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 6253)
  AUTHORS   Ma H, McLean JR, Chao LF, Mana-Capelli S, Paramasivam M, Hagstrom 
            KA, Gould KL, McCollum D
  TITLE     A highly efficient multifunctional tandem affinity purification 
            approach applicable to diverse organisms.
  JOURNAL   Mol Cell Proteomics. 2012 Aug;11(8):501-11. doi: 
            10.1074/mcp.O111.016246. Epub 2012 Apr 3.
  PUBMED    22474084
REFERENCE   2  (bases 1 to 6253)
  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/mcp.O111.016246"; journalName: "Mol Cell Proteomics"; date:
            "2012-08"; volume: "11"; issue: "8"; pages: "501-11"
COMMENT     Sequence Label: pcDNA4-TO-Puromycin-mVenus-MAP
FEATURES             Location/Qualifiers
     source          1..6253
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(43..62)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        234..613
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        614..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     768..788
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    819..837
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    840..858
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     868..892
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(1061..1063,1064..1066,1067..1579)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIE"
     primer_bind     complement(1106..1127)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1367..1386)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             1601..1624
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             1634..1747
                     /codon_start=1
                     /product="streptavidin-binding peptide"
                     /label=SBP
                     /note="selected from a peptide library; binds streptavidin 
                     with nanomolar affinity (Keefe et al., 2001)"
                     /translation="MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP"
     CDS             1766..1789
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2037..2054)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2043..2267
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2313..2741
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2400..2419)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2610..2631
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2750..2770)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2768..3065
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2935..3070
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3200..3799
                     /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(3200..3219)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3696..3716
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3929..4050
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3966..3985)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4020..4039
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4099..4115)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4099..4115)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4112..4134)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4123..4139
                     /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(4147..4153,4154..4171,4172..4177))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4192..4213
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4330..4347)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4501..5086)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4581..4600)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5257..6048,6049..6117))
                     /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     5880..5899
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6118..6222)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
       61 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
      121 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
      181 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg
      241 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
      301 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
      361 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
      421 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
      481 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
      541 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
      601 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
      661 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggaacca
      721 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
      781 taggcgtgta cggtgggagg tctatataag cagagctctc cctatcagtg atagagatct
      841 ccctatcagt gatagagatc gtcgacgagc tcgtttagtg aaccgtcaga tcgcctggag
      901 acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca gcctccggac
      961 tctagcgttt aaacttaagc ttggtaccga gctcggatcc actagtccag tgtggtggaa
     1021 ttctgcagat atccagcaca gtggcggccg cggcagcagc atggtgagca agggcgagga
     1081 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     1141 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagct
     1201 gatctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgggcta
     1261 cggcctgcag tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     1321 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     1381 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     1441 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     1501 cagccacaac gtctatatca ccgccgacaa gcagaagaac ggcatcaagg ccaacttcaa
     1561 gatccgccac aacatcgagg acggcagcct cgagtccgct catcaccatc accatcacca
     1621 tcacggcagc gccatggacg agaagaccac cggctggcgg ggcggccacg tggtggaggg
     1681 cctggccggc gagctggagc agctgcgggc caggctggag caccaccctc agggccagcg
     1741 ggagccctcc ggcggcggta gcgctgacta caaagacgat gacgacaagg gcagcgctag
     1801 cagcggcggc ggcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     1861 ccccgtgctg ctgcccgaca accactacct gagctaccag tccaagctga gcaaagaccc
     1921 caacgagaag cgcgatcaca tggtcctgaa ggagcgcgtg accgccgccg ggatcactct
     1981 cggcatggac gagctgtaca agtaatctag agggcccgtt taaacccgct gatcagcctc
     2041 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     2101 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     2161 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     2221 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     2281 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     2341 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     2401 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     2461 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     2521 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     2581 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     2641 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     2701 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     2761 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     2821 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     2881 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     2941 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     3001 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     3061 tttggaggcc taggaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg
     3121 aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt
     3181 cggatctgat cagcacgtga tgaccgagta caagcccacg gtgcgcctcg ccacccgcga
     3241 cgacgtcccc cgggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg
     3301 ccacaccgtc gacccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct
     3361 cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc
     3421 ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg
     3481 catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc
     3541 gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca
     3601 ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc
     3661 cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct
     3721 cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac
     3781 ccgcaagccc ggtgcctgac acgtgctacg agatttcgat tccaccgccg ccttctatga
     3841 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     3901 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     3961 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     4021 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     4081 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     4141 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     4201 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     4261 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     4321 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     4381 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     4441 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     4501 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     4561 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     4621 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     4681 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     4741 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     4801 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     4861 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     4921 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     4981 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggttt
     5041 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5101 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     5161 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     5221 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     5281 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     5341 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     5401 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     5461 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     5521 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     5581 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     5641 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     5701 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     5761 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     5821 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     5881 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     5941 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     6001 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6061 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     6121 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     6181 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     6241 gccacctgac gtc
//
