LOCUS       sequence-158473-        6748 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac for EOS-GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6748)
  AUTHORS   Takashima Y, Guo G, Loos R, Nichols J, Ficz G, Krueger F, Oxley D, 
            Santos F, Clarke J, Mansfield W, Reik W, Bertone P, Smith A
  TITLE     Resetting Transcription Factor Control Circuitry toward Ground-State
            Pluripotency in Human.
  JOURNAL   Cell. 2014 Sep 11;158(6):1254-69. doi: 10.1016/j.cell.2014.08.029.
  PUBMED    25215486
REFERENCE   2  (bases 1 to 6748)
  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: 
            "10.1016/j.cell.2014.08"; journalName: "Cell"; date: "2014-09-11- 
            11"; volume: "158"; issue: "6"; pages: "1254-69"
COMMENT     Sequence Label: pPB-EOS-GFP-IP
FEATURES             Location/Qualifiers
     source          1..6748
                     /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"
     repeat_region   664..698
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    973..1006
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    1045..1269
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1258..1275
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(1356..1955)
                     /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(1439..1459)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     1936..1955
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     misc_feature    join(1956..1964,1965..1967,1968..2541)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2128..2147)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     2357..2374
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             complement(join(2608..3321,3322..3324,3325..3327))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(2611..3321,3322..3324,3325..3327))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2653..2674)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3002..3021
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3261..3282
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      3324..3333
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(4230..4246)
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     protein_bind    4270..4303
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     repeat_region   4474..4536
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4560..4580)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4560..4578)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4599..4615)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4599..4615)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4612..4634)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4623..4639
                     /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(4647..4653,4654..4671,4672..4677))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4692..4713
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4830..4847)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5001..5589)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5081..5100)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5760..6551,6552..6620))
                     /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     6383..6402
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6621..6725)
                     /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 tggagctctt
      661 taaccctaga aagatagtct gcgtaaaatt gacgcatgca ttcttgaaat attgctctct
      721 ctttctaaat agcgcgaatc cgtcgctgtg catttaggac atctcagtcg ccgcttggag
      781 ctcccgtgag gcgtgcttgt caatgcggta agtgtcactg attttgaact ataacgaccg
      841 cgtgagtcaa aatgacgcat gattatcttt tacgtgactt ttaagattta actcatacga
      901 taattatatt gttatttcat gttctactta cgtgataact tattatatat atattttctt
      961 gttatagata tcataacttc gtataatgta tgctatacga agttatgcgg ccctcgaggc
     1021 tgcaggttct ttccgcctca gaagccatag agcccaccgc atccccagca tgcctgctat
     1081 tgtcttccca atcctccccc ttgctgtcct gccccacccc accccccaga atagaatgac
     1141 acctactcag acaatgcgat gcaatttcct cattttatta ggaaaggaca gtgggagtgg
     1201 caccttccag ggtcaaggaa ggcacggggg aggggcaaac aacagatggc tggcaactag
     1261 aaggcacagt cgaggctgat cagcgagctc tagatcatcg atgcatgggg tcgtgcgctc
     1321 ctttcggtcg ggcgctgcgg gtcgtggggc gggcgtcagg caccgggctt gcgggtcatg
     1381 caccaggtgc gcggtccttc gggcacctcg acgtcggcgg tgacggtgaa gccgagccgc
     1441 tcgtagaagg ggaggttgcg gggcgcggag gtctccagga aggcgggcac cccggcgcgc
     1501 tcggccgcct ccactccggg gagcacgacg gcgctgccca gacccttgcc ctggtggtcg
     1561 ggcgagacgc cgacggtggc caggaaccac gcgggctcct tgggccggtg cggcgccagg
     1621 aggccttcca tctgttgctg cgcggccagc cgggaaccgc tcaactcggc catgcgcggg
     1681 ccgatctcgg cgaacaccgc ccccgcttcg acgctctccg gcgtggtcca gaccgccacc
     1741 gcggcgccgt cgtccgcgac ccacaccttg ccgatgtcga gcccgacgcg cgtgaggaag
     1801 agttcttgca gctcggtgac ccgctcgatg tggcggtccg gatcgacggt gtggcgcgtg
     1861 gcggggtagt cggcgaacgc ggcggcgagg gtgcgtacgg cccgggggac gtcgtcgcgg
     1921 gtggcgaggc gcaccgtggg cttgtactcg gtcatggttg tggccatatt atcatcgtgt
     1981 ttttcaaagg aaaaccacgt ccccgtggtt cggggggcct agacgttttt taacctcgac
     2041 taaacacatg taaagcatgt gcaccgaggc cccagatcag atcccataca atggggtacc
     2101 ttctgggcat ccttcagccc cttgttgaat acgcttgagg agagccattt gactctttcc
     2161 acaactatcc aactcacaac gtggcactgg ggttgtgccg cctttgcagg tgtatcttat
     2221 acacgtggct tttggccgca gaggcacctg tcgccaggtg gggggttccg ctgcctgcaa
     2281 agggtcgcta cagacgttgt ttgtcttcaa gaagcttcca gaggaactgc ttccttcacg
     2341 acattcaaca gaccttgcat tcctttggcg agaggggaaa gacccctagg aatgctcgtc
     2401 aagaagacag ggccaggttt ccgggccctc acattgccaa aagacggcaa tatggtggaa
     2461 aataacatat agacaaacgc acaccggcct tattccaagc ggcttcggcc agtaacgtta
     2521 gggggggggg agggagaggg gcggaattcg atatcaagct tatcgagcgg ccgctcgata
     2581 ccgtcgatct cagtggacta gtgctagcta cttgtacagc tcgtccatgc cgagagtgat
     2641 cccggcggcg gtcacgaact ccagcaggac catgtgatcg cgcttctcgt tggggtcttt
     2701 gctcagggcg gactgggtgc tcaggtagtg gttgtcgggc agcagcacgg ggccgtcgcc
     2761 gatgggggtg ttctgctggt agtggtcggc gagctgcacg ctgccgtcct cgatgttgtg
     2821 gcggatcttg aagttcacct tgatgccgtt cttctgcttg tcggccatga tatagacgtt
     2881 gtggctgttg tagttgtact ccagcttgtg ccccaggatg ttgccgtcct ccttgaagtc
     2941 gatgcccttc agctcgatgc ggttcaccag ggtgtcgccc tcgaacttca cctcggcgcg
     3001 ggtcttgtag ttgccgtcgt ccttgaagaa gatggtgcgc tcctggacgt agccttcggg
     3061 catggcggac ttgaagaagt cgtgctgctt catgtggtcg gggtagcggc tgaagcactg
     3121 cacgccgtag gtcagggtgg tcacgagggt gggccagggc acgggcagct tgccggtggt
     3181 gcagatgaac ttcagggtca gcttgccgta ggtggcatcg ccctcgccct cgccggacac
     3241 gctgaacttg tggccgttta cgtcgccgtc cagctcgacc aggatgggca ccaccccggt
     3301 gaacagctcc tcgcccttgc tcaccatggt ggctttacca acagtaccgg aatgccaagc
     3361 ttacttagat cgcagatctt gttgcggccg ccagcagctc gcaacgtgaa cggttcgact
     3421 gagaaggccg ctcgagcttt aagagaggaa tctagacggg gcggaagaaa gaatgtagcc
     3481 aagacagtta ctctgatcaa ggctcaaatt ttatagttgc gacactagtt atgaaggaag
     3541 ggggagggga cccgattccc gccgaataat ctctggtcca gtagaaaggt gcacgggtgt
     3601 ggctccgcag gttccagcag tgggcgtggc agaacgaatg agcaggaagc tccacccctg
     3661 agcaagcagg tttcaggctg ggggagggga gaattcacta gtgattaagc tcgagctagg
     3721 acgagaggga cccctcccca accatcttct ctgcccccag ggcagagcta tcatgcacct
     3781 ttgttatgca tctgccgtct gcccagaact ctcaacctcc ctggggggag gggcagggtc
     3841 gggctacaac ctccccacac ccgaattcac tagtgattaa gctcgagcta ggacgagagg
     3901 gacccctccc caaccatctt ctctgccccc agggcagagc tatcatgcac ctttgttatg
     3961 catctgccgt ctgcccagaa ctctcaacct ccctgggggg aggggcaggg tcgggctaca
     4021 acctccccac acccgaattc actagtgatt aagctcgagc taggacgaga gggacccctc
     4081 cccaaccatc ttctctgccc ccagggcaga gctatcatgc acctttgtta tgcatctgcc
     4141 gtctgcccag aactctcaac ctccctgggg ggaggggcag ggtcgggcta caacctcccc
     4201 acacccgaat tccggcgcct agagaaggag tgagggctgg ataaagggag gatccgcggc
     4261 cgcgtcgaga taacttcgta taatgtatgc tatacgaagt tatgttttgt tactttatag
     4321 aagaaatttt gagtttttgt ttttttttaa taaataaata aacataaata aattgtttgt
     4381 tgaatttatt attagtatgt aagtgtaaat ataataaaac ttaatatcta ttcaaattaa
     4441 taaataaacc tcgatataca gaccgataaa acacatgcgt caattttacg catgattatc
     4501 tttaacgtac gtcacaatat gattatcttt ctagggttaa agagctccag cttttgttcc
     4561 ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga
     4621 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     4681 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     4741 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4801 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4861 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4921 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4981 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5041 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5101 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5161 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5221 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5281 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5341 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5401 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     5461 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5521 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5581 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5641 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5701 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5761 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5821 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5881 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5941 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     6001 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     6061 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     6121 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6181 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6241 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6301 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6361 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6421 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6481 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6541 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6601 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6661 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6721 ccgcgcacat ttccccgaaa agtgccac
//
