LOCUS       sequence-440524-        6241 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Flp recombinase and puromycin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6241)
  AUTHORS   Waite JB, Boytz R, Traeger AR, Lind TM, Lumbao-Conradson K, Torigoe 
            SE
  TITLE     A suboptimal OCT4-SOX2 binding site facilitates the naive-state 
            specific function of a Klf4 enhancer.
  JOURNAL   PLoS One. 2024 Sep 30;19(9):e0311120. doi: 
            10.1371/journal.pone.0311120. eCollection 2024.
  PUBMED    39348365
REFERENCE   2  (bases 1 to 6241)
  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: "PLoS One.";
            date: "2024-09-30"; volume: "19(9):e0311120. doi"; pages: " 
            10.1371/journal.pone.0311120. eCollection 2024"
COMMENT     Sequence Label: pBSII-PGK-FLPo-IRES-Puro
FEATURES             Location/Qualifiers
     source          1..6241
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        659..1158
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(694..717)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1091..1110
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     1175..1191
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1176..1192
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             join(1213..1218,1219..1239,1240..2511)
                     /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"
     misc_feature    2521..3094
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    2544..3096
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2688..2705)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2915..2934
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3111..3710
                     /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(3111..3130)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3607..3627
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(3776..3796)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    3838..3959
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3875..3894)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3929..3948
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4053..4073)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4053..4071)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4092..4108)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4092..4108)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4105..4127)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4116..4132
                     /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(4140..4146,4147..4164,4165..4170))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4185..4206
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4323..4340)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4494..5082)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4574..4593)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5253..6044,6045..6113))
                     /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     5876..5895
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6114..6218)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg
      661 gtaggggagg cgcttttccc aaggcagtct ggagcatgcg ctttagcagc cccgctgggc
      721 acttggcgct acacaagtgg cctctggcct cgcacacatt ccacatccac cggtaggcgc
      781 caaccggctc cgttctttgg tggccccttc gcgccacctt ctactcctcc cctagtcagg
      841 aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt
      901 ctcactagtc tcgtgcagat ggacagcacc gctgagcaat ggaagcgggt aggcctttgg
      961 ggcagcggcc aatagcagct ttgctccttc gctttctggg ctcagaggct gggaaggggt
     1021 gggtccgggg gcgggctcag gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc
     1081 ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc
     1141 tcatctccgg gcctttcgac catcgccgcc accctcgagg tcgacggtat cgataagctt
     1201 gatatcgaat tcatggctcc taagaagaag aggaaggtga tgagccagtt cgacatcctg
     1261 tgcaagaccc cccccaaggt gctggtgcgg cagttcgtgg agagattcga gaggcccagc
     1321 ggcgagaaga tcgccagctg tgccgccgag ctgacctacc tgtgctggat gatcacccac
     1381 aacggcaccg ccatcaagag ggccaccttc atgagctaca acaccatcat cagcaacagc
     1441 ctgagcttcg acatcgtgaa caagagcctg cagttcaagt acaagaccca gaaggccacc
     1501 atcctggagg ccagcctgaa gaagctgatc cccgcctggg agttcaccat catcccttac
     1561 aacggccaga agcaccagag cgacatcacc gacatcgtgt ccagcctgca gctgcagttc
     1621 gagagcagcg aggaggccga caagggcaac agccacagca agaagatgct gaaggccctg
     1681 ctgtccgagg gcgagagcat ctgggagatc accgagaaga tcctgaacag cttcgagtac
     1741 accagcaggt tcaccaagac caagaccctg taccagttcc tgttcctggc cacattcatc
     1801 aactgcggca ggttcagcga catcaagaac gtggacccca agagcttcaa gctggtgcag
     1861 aacaagtacc tgggcgtgat cattcagtgc ctggtgaccg agaccaagac aagcgtgtcc
     1921 aggcacatct actttttcag cgccagaggc aggatcgacc ccctggtgta cctggacgag
     1981 ttcctgagga acagcgagcc cgtgctgaag agagtgaaca ggaccggcaa cagcagcagc
     2041 aacaagcagg agtaccagct gctgaaggac aacctggtgc gcagctacaa caaggccctg
     2101 aagaagaacg ccccctaccc catcttcgct atcaagaacg gccctaagag ccacatcggc
     2161 aggcacctga tgaccagctt tctgagcatg aagggcctga ccgagctgac aaacgtggtg
     2221 ggcaactgga gcgacaagag ggcctccgcc gtggccagga ccacctacac ccaccagatc
     2281 accgccatcc ccgaccacta cttcgccctg gtgtccaggt actacgccta cgaccccatc
     2341 agcaaggaga tgatcgccct gaaggacgag accaacccca tcgaggagtg gcagcacatc
     2401 gagcagctga agggcagcgc cgagggcagc atcagatacc ccgcctggaa cggcatcatc
     2461 agccaggagg tgctggacta cctgagcagc tacatcaaca ggcggatctg agcggccgca
     2521 cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg
     2581 tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg
     2641 gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg
     2701 aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca
     2761 aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct
     2821 ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca
     2881 cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa
     2941 ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg
     3001 cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg
     3061 ggacgtggtt ttcctttgaa aaacacgatg ataatatggc cacacatatg atgaccgagt
     3121 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg
     3181 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg
     3241 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg
     3301 tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag
     3361 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg
     3421 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt
     3481 tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg
     3541 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg
     3601 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg
     3661 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga atcgatgtcg
     3721 acaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     3781 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgcg ttaactaaac
     3841 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     3901 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     3961 catgtctgga attgactcaa atgatgtcaa ttagtctatc agaagctcat ctggtctccc
     4021 ttccggggga caaggagctc cagcttttgt tccctttagt gagggttaat tgcgcgcttg
     4081 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     4141 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     4201 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4261 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     4321 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     4381 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     4441 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     4501 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     4561 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     4621 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     4681 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     4741 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     4801 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     4861 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     4921 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     4981 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     5041 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5101 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5161 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5221 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5281 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     5341 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     5401 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     5461 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     5521 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     5581 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     5641 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     5701 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     5761 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     5821 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     5881 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     5941 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6001 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6061 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6121 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6181 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6241 c
//
