LOCUS       sequence-471572-        4202 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty mammalian expression plasmid for cloning N-terminally 
            HA-tagged proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4202)
  AUTHORS   Addgene Research Program
  TITLE     Addgene empty and control mammalian expression backbones
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4202)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pAG CMV N-HA Puro
FEATURES             Location/Qualifiers
     source          1..4202
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     100..122
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     114..131
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     115..131
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     141..160
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        141..159
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     183..199
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     184..200
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        218..421
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     372..392
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             475..501
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             571..627
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             628..1227
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTLTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(628..647)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1124..1144
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1311..1432
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1348..1367)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1402..1421
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1529..1549)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1529..1547)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1568..1584)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1568..1584)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1581..1603)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1592..1608
                     /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(1616..1622,1623..1640,1641..1646))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1661..1682
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1799..1816)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1970..2558)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2050..2069)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2729..3520,3521..3589))
                     /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     3352..3371
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3590..3694)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3720..4175)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3857..3878)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4069..4088
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
       61 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
      121 cgacggccag tgagcgcgcg taatacgact cactataggg cgaatgggta ccggaccccc
      181 cctcgaggtc gacggtatcg ggggcatgct taattaagtg atgcggtttt ggcagtacat
      241 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      301 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
      361 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      421 ttctagaggt ttagtgaacc gtcagatccg ctagcgctac cggtcgccac catgtaccca
      481 tacgatgttc cggattatgc tttcgaaggc gcgccgcgat cgcgggcccg atatctgatc
      541 actcgagggc cggccatgca tggatccggc gcaacaaact tctctctgct gaaacaagcc
      601 ggagatgtcg aagagaatcc tggaccgatg accgagtaca agcccacggt gcgcctcgcc
      661 acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc
      721 gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa
      781 ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgca
      841 gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc
      901 ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc
      961 ctcctggcac cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg
     1021 cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc
     1081 gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac
     1141 gagcggctcg gcttcaccct caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg
     1201 tgcatgaccc gcaagcccgg tgcctgaacg cgtgtagtag ttcaagtcat cttattattc
     1261 agtatttata acttgcaaag aaatgaatat cagagagtga gaggtgtaca aacttgttta
     1321 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     1381 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct taactagtgg
     1441 cgccaagccg aattctgcag atatccatca cactggcggc cgctcgacta gagcggccgc
     1501 caccgcggtg gagctccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt
     1561 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     1621 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     1681 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     1741 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     1801 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     1861 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     1921 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     1981 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     2041 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     2101 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     2161 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     2221 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     2281 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     2341 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     2401 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     2461 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     2521 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     2581 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     2641 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     2701 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     2761 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     2821 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     2881 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     2941 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     3001 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     3061 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     3121 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     3181 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     3241 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     3301 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     3361 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     3421 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     3481 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     3541 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     3601 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     3661 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta
     3721 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     3781 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     3841 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     3901 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     3961 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     4021 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     4081 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     4141 acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa
     4201 ct
//
