LOCUS       sequence-247151-        6716 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Puromycin resistance gene and FLP recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6716)
  AUTHORS   Nam HS, Capecchi MR
  TITLE     2A plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6716)
  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            "Unpublished"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: pCAGGS-pac-T2A-FLPo
FEATURES             Location/Qualifiers
     source          1..6716
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        11..390
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        392..669
                     /label=chicken beta-actin promoter
     intron          670..1686
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1610..1632
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1694..1713
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1744..2340
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MAEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     2240..2260
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2362..2415
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(2431..2436,2437..2457,2458..3729)
                     /codon_start=1
                     /product="nuclear-targeted site-specific recombinase"
                     /label=FLPo
                     /note="FLPo is a mouse codon-optimized fusion between the 
                     SV40 nuclear localization signal and a thermostable version
                     of the Saccharomyces cerevisiae site-specific recombinase 
                     FLP.  Recombination occurs at FRT sequences."
                     /translation="MAPKKKRKVMSQFDILCKTPPKVLVRQFVERFERPSGEKIASCAA
                     ELTYLCWMITHNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKK
                     LIPAWEFTIIPYNGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESI
                     WEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGV
                     IIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEY
                     QLLKDNLVRSYNKALKKNAPYPIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWS
                     DKRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQL
                     KGSAEGSIRYPAWNGIISQEVLDYLSSYINRRI"
     primer_bind     complement(3744..3763)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3809..3864
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3863..3882)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4225..4241)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4225..4241)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4238..4260)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4249..4265
                     /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(4273..4279,4280..4297,4298..4303))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4318..4339
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4398..4593
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4444..4579
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4599..4733
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4636..4655)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4690..4709
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4801..4818)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4972..5560)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5052..5071)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5731..6522,6523..6591))
                     /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     6354..6373
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6592..6696)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacctgggtc gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
       61 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
      121 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
      181 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
      241 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
      301 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
      361 atctacgtat tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact
      421 ctccccatct cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt
      481 tgtgcagcga tgggggcggg gggggggggg gggcgcgcgc caggcggggc ggggcggggc
      541 gaggggcggg gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc
      601 cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg
      661 cggcgggcgg gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg
      721 cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc
      781 ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg
      841 ctgcgtgaaa gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg
      901 ggtgcgtgcg tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc
      961 tgtgagcgct gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc
     1021 gcggccgggg gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg
     1081 cggggtgtgt gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc
     1141 cccctgcacc cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt
     1201 acggggcgtg gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg
     1261 ggcggggcgg ggccgcctcg ggccggggag ggctcggggg aggggcgcgg cggcccccgg
     1321 agcgccggcg gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc
     1381 gagagggcgc agggacttcc tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc
     1441 cgccgcaccc cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg
     1501 ggcggggagg gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg
     1561 ggctgtccgc ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc
     1621 tggcgtgtga ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc
     1681 ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattc
     1741 gccatggccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc
     1801 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg
     1861 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     1921 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     1981 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     2041 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag
     2101 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg
     2161 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc
     2221 ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     2281 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     2341 ggcggctccg gcggccgctc cgagggccgc ggctccctgc tgacctgcgg cgacgtggag
     2401 gagaaccccg gccccgcccc cggctccacc atggctccta agaagaagag gaaggtgatg
     2461 agccagttcg acatcctgtg caagaccccc cccaaggtgc tggtgcggca gttcgtggag
     2521 agattcgaga ggcccagcgg cgagaagatc gccagctgtg ccgccgagct gacctacctg
     2581 tgctggatga tcacccacaa cggcaccgcc atcaagaggg ccaccttcat gagctacaac
     2641 accatcatca gcaacagcct gagcttcgac atcgtgaaca agagcctgca gttcaagtac
     2701 aagacccaga aggccaccat cctggaggcc agcctgaaga agctgatccc cgcctgggag
     2761 ttcaccatca tcccttacaa cggccagaag caccagagcg acatcaccga catcgtgtcc
     2821 agcctgcagc tgcagttcga gagcagcgag gaggccgaca agggcaacag ccacagcaag
     2881 aagatgctga aggccctgct gtccgagggc gagagcatct gggagatcac cgagaagatc
     2941 ctgaacagct tcgagtacac cagcaggttc accaagacca agaccctgta ccagttcctg
     3001 ttcctggcca cattcatcaa ctgcggcagg ttcagcgaca tcaagaacgt ggaccccaag
     3061 agcttcaagc tggtgcagaa caagtacctg ggcgtgatca ttcagtgcct ggtgaccgag
     3121 accaagacaa gcgtgtccag gcacatctac tttttcagcg ccagaggcag gatcgacccc
     3181 ctggtgtacc tggacgagtt cctgaggaac agcgagcccg tgctgaagag agtgaacagg
     3241 accggcaaca gcagcagcaa caagcaggag taccagctgc tgaaggacaa cctggtgcgc
     3301 agctacaaca aggccctgaa gaagaacgcc ccctacccca tcttcgctat caagaacggc
     3361 cctaagagcc acatcggcag gcacctgatg accagctttc tgagcatgaa gggcctgacc
     3421 gagctgacaa acgtggtggg caactggagc gacaagaggg cctccgccgt ggccaggacc
     3481 acctacaccc accagatcac cgccatcccc gaccactact tcgccctggt gtccaggtac
     3541 tacgcctacg accccatcag caaggagatg atcgccctga aggacgagac caaccccatc
     3601 gaggagtggc agcacatcga gcagctgaag ggcagcgccg agggcagcat cagatacccc
     3661 gcctggaacg gcatcatcag ccaggaggtg ctggactacc tgagcagcta catcaacagg
     3721 cggatctgat aaggcgcgcc ggatcttttt ccctctgcca aaaattatgg ggacatcatg
     3781 aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg
     3841 tgttggaatt ttttgtgtct ctcactcgga aggacatatg ggagggcaaa tcatttaaaa
     3901 catcagaatg agtatttggt ttagagtttg gcaacatatg cccatatgct ggctgccatg
     3961 aacaaaggtt ggctataaag aggtcatcag tatatgaaac agccccctgc tgtccattcc
     4021 ttattccata gaaaagcctt gacttgaggt tagatttttt ttatattttg ttttgtgtta
     4081 tttttttctt taacatccct aaaattttcc ttacatgttt tactagccag atttttcctc
     4141 ctctcctgac tactcccagt catagctgtc cctcttctct tatggagatc cctcgacctg
     4201 cagcccaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     4261 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     4321 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     4381 gtcgtgccag cggatccgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     4441 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     4501 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     4561 ggaggctttt ttggaggcct aggcttttgc aaaaagctaa cttgtttatt gcagcttata
     4621 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     4681 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg atccgctgca
     4741 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     4801 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4861 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     4921 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     4981 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5041 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5101 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5161 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5221 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5281 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5341 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5401 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5461 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5521 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5581 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     5641 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5701 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     5761 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     5821 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     5881 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     5941 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6001 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     6061 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     6121 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     6181 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     6241 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6301 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6361 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6421 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6481 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6541 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     6601 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     6661 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgc
//
