LOCUS       sequence-469913-        4199 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty mammalian expression plasmid for cloning C-terminally 
            HA-tagged proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4199)
  AUTHORS   Addgene Research Program
  TITLE     Addgene empty and control mammalian expression backbones
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4199)
  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            "Unpublished"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 C-HA Puro
FEATURES             Location/Qualifiers
     source          1..4199
                     /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             532..558
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             568..624
                     /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             625..1224
                     /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(625..644)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1121..1141
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1308..1429
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1345..1364)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1399..1418
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1526..1546)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1526..1544)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1565..1581)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1565..1581)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1578..1600)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1589..1605
                     /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(1613..1619,1620..1637,1638..1643))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1658..1679
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1796..1813)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1967..2555)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2047..2066)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2726..3517,3518..3586))
                     /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     3349..3368
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3587..3691)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3717..4172)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3854..3875)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4066..4085
                     /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 cttcgaaggc
      481 gcgccgcgat cgcgatatca tgcattgatc actcgagggc cggccgggcc ctacccatac
      541 gatgttccgg attacgctgg atccggcgca acaaacttct ctctgctgaa acaagccgga
      601 gatgtcgaag agaatcctgg accgatgacc gagtacaagc ccacggtgcg cctcgccacc
      661 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
      721 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
      781 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgcagcg
      841 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
      901 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
      961 ctggcaccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc
     1021 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     1081 cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag
     1141 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     1201 atgacccgca agcccggtgc ctgaacgcgt gtagtagttc aagtcatctt attattcagt
     1261 atttataact tgcaaagaaa tgaatatcag agagtgagag gtgtacaaac ttgtttattg
     1321 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     1381 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ctagtggcgc
     1441 caagccgaat tctgcagata tccatcacac tggcggccgc tcgactagag cggccgccac
     1501 cgcggtggag ctccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat
     1561 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     1621 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     1681 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     1741 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     1801 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     1861 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     1921 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     1981 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     2041 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     2101 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     2161 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     2221 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     2281 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     2341 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     2401 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     2461 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     2521 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     2581 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     2641 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     2701 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     2761 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     2821 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     2881 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     2941 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     3001 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     3061 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     3121 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     3181 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     3241 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     3301 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     3361 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     3421 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     3481 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     3541 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     3601 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     3661 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat
     3721 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     3781 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     3841 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     3901 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     3961 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     4021 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     4081 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     4141 cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc tgcgcaact
//
