LOCUS       sequence-60940-e        6609 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 6609)
  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 6609)
  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-Hygromycin-sfGFP-MAP
FEATURES             Location/Qualifiers
     source          1..6609
                     /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             1598..1621
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             1631..1744
                     /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             1763..1786
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2034..2051)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2040..2264
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2310..2738
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2397..2416)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2607..2628
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2747..2767)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2752..3081
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2932..3067
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2994..3013
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3130..4155
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3130..4155
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    4285..4406
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4322..4341)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4376..4395
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4455..4471)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4455..4471)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4468..4490)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4479..4495
                     /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(4503..4509,4510..4527,4528..4533))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4548..4569
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4686..4703)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4857..5442)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4937..4956)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5613..6404,6405..6473))
                     /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     6236..6255
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6474..6578)
                     /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 cggcagcgca atgcgtaaag gcgaagagct
     1081 gttcactggt gtcgtcccta ttctggtgga actggatggt gatgtcaacg gtcataagtt
     1141 ttccgtgcgt ggcgagggtg aaggtgacgc aactaatggt aaactgacgc tgaagttcat
     1201 ctgtactact ggtaaactgc cggtaccttg gccgactctg gtaacgacgc tgacttatgg
     1261 tgttcagtgc tttgctcgtt atccggacca tatgaagcag catgacttct tcaagtccgc
     1321 catgccggaa ggctatgtgc aggaacgcac gatttccttt aaggatgacg gcacgtacaa
     1381 aacgcgtgcg gaagtgaaat ttgaaggcga taccctggta aaccgcattg agctgaaagg
     1441 cattgacttt aaagaagacg gcaatatcct gggccataag ctggaataca attttaacag
     1501 ccacaatgtt tacatcaccg ccgataaaca aaaaaatggc attaaagcga attttaaaat
     1561 tcgccacaac gtggaggatg gcagcctcga gtccgctcat caccatcacc atcaccatca
     1621 cggcagcgcc atggacgaga agaccaccgg ctggcggggc ggccacgtgg tggagggcct
     1681 ggccggcgag ctggagcagc tgcgggccag gctggagcac caccctcagg gccagcggga
     1741 gccctccggc ggcggtagcg ctgactacaa agacgatgac gacaagggca gcgctagcag
     1801 cggcggcagc gtgcagctgg ctgatcacta ccagcaaaac actccaatcg gtgatggtcc
     1861 tgttctgctg ccagacaatc actatctgag cacgcaaagc gttctgtcta aagatccgaa
     1921 cgagaaacgc gatcatatgg ttctgctgga gttcgtaacc gcagcgggca tcacgcatgg
     1981 tatggatgaa ctgtacaaat gatctagagg gcccgtttaa acccgctgat cagcctcgac
     2041 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     2101 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     2161 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     2221 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     2281 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     2341 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     2401 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     2461 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     2521 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     2581 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     2641 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     2701 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     2761 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2821 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     2881 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     2941 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     3001 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     3061 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     3121 cagcacgtga tgaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa
     3181 aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc
     3241 agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc
     3301 tacaaagatc gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg
     3361 cttgacattg gggaattcag cgagagcctg acctattgca tctcccgccg tgcacagggt
     3421 gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag
     3481 gccatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga
     3541 ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc
     3601 catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct
     3661 ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg
     3721 gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg
     3781 agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg
     3841 tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca
     3901 ggatcgccgc ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc
     3961 ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc
     4021 cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg
     4081 accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg
     4141 agggcaaagg aatagcacgt gctacgagat ttcgattcca ccgccgcctt ctatgaaagg
     4201 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     4261 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     4321 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     4381 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     4441 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     4501 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     4561 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4621 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     4681 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4741 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4801 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4861 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4921 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4981 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     5041 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     5101 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     5161 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     5221 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     5281 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     5341 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggttttttt
     5401 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5461 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5521 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5581 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5641 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     5701 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     5761 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     5821 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     5881 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     5941 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     6001 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     6061 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     6121 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6181 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     6241 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6301 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6361 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6421 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6481 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6541 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6601 cctgacgtc
//
