LOCUS       sequence-425304-        6920 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An empty backbone for cloning of homology arms; ITR-flanked, 
            EF1alpha-driven expression of puromycin-EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6920)
  AUTHORS   Zhao Y, Pan Z, Hong Z, Sun M, Hong Y, Peng X, Li X, Wang X, Wang K
  TITLE     Protocol for scarless genome editing of human pluripotent stem cell 
            based on orthogonal selective reporters.
  JOURNAL   STAR Protoc. 2024 May 23;5(2):103084. doi: 
            10.1016/j.xpro.2024.103084.
  PUBMED    38787727
REFERENCE   2  (bases 1 to 6920)
  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: "STAR 
            Protoc."; date: "2024-05-23"; volume: "5(2):103084. doi"; pages: " 
            10.1016/j.xpro.2024.103084"
COMMENT     Sequence Label: P2A-NLS-tdtomato-EPG backbone
FEATURES             Location/Qualifiers
     source          1..6920
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(37..54)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    43..267
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   458..520
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     rep_origin      complement(599..1187)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(679..698)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1358..2149,2150..2218))
                     /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     1981..2000
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2219..2323)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2391..2409
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             2442..2498
                     /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             2508..2528
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2538..2558
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2565..3995
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGRYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     repeat_region   4007..4041
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    4331..4351
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        4352..5530
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          4583..5521
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     5478..5498
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             5540..6136
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APHNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(5540..5559)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             6143..6199
                     /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             join(6200..6202,6203..6205,6206..6919)
                     /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             join(6200..6202,6203..6205,6206..6916)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6245..6266)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6506..6525)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6853..6874
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 tcgagtctag agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
       61 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
      121 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
      181 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
      241 atgctgggga tgcggtgggc tctatgggcg gccctgtccc tcataaaagt tttgttactt
      301 tatagaagaa attttgagtt tttgtttttt ttaataaata aataaacata aataaattgt
      361 ttgttgaatt tattattagt atgtaagtgt aaatataata aaacttaata tctattcaaa
      421 ttaataaata aacctcgata tacagaccga taaaacacat gcgtcaattt tacacatgat
      481 tatctttaac gtacgtcaca atatgattat ctttctaggg ttaactctct ctgaattcca
      541 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
      601 tccataggct ccgcccccct gacgagcatc acgaaaatcg acgctcaagt cagaggtggc
      661 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
      721 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
      781 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
      841 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
      901 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
      961 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     1021 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     1081 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     1141 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     1201 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     1261 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     1321 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     1381 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     1441 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     1501 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     1561 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     1621 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     1681 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     1741 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     1801 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     1861 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     1921 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     1981 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     2041 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     2101 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     2161 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     2221 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     2281 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     2341 tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta
     2401 tcacgaggcc ctttcgtcgg cgcgggtacc ggggatccgt cgcgacgaat tttagtctac
     2461 tgaaacaagc gggagacgtg gaggaaaacc ctggacctat cgatggtcca aaaaagaaga
     2521 gaaaggtatt cgatggtcca aaaaagaaga gaaaggtatt cgatatggtg agcaagggcg
     2581 aggaggtcat caaagagttc atgcgcttca aggtgcgcat ggagggctcc atgaacggcc
     2641 acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca
     2701 agctgaaggt gaccaagggc ggccccctgc ccttcgcctg ggacatcctg tccccccagt
     2761 tcatgtacgg ctccaaggcg tacgtgaagc accccgccga catccccgat tacaagaagc
     2821 tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggtctgg
     2881 tgaccgtgac ccaggactcc tccctgcagg acggcacgct gatctacaag gtgaagatgc
     2941 gcggcaccaa cttccccccc gacggccccg taatgcagaa gaagaccatg ggctgggagg
     3001 cctccaccga gcgcctgtac ccccgcgacg gcgtgctgaa gggcgagatc caccaggccc
     3061 tgaagctgaa ggacggcggc cactacctgg tggagttcaa gaccatctac atggccaaga
     3121 agcccgtgca actgcccggc tactactacg tggacaccaa gctggacatc acctcccaca
     3181 acgaggacta caccatcgtg gaacagtacg agcgctccga gggccgccac cacctgttcc
     3241 tggggcatgg caccggcagc accggcagcg gcagctccgg caccgcctcc tccgaggaca
     3301 acaacatggc cgtcatcaaa gagttcatgc gcttcaaggt gcgcatggag ggctccatga
     3361 acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga
     3421 ccgccaagct gaaggtgacc aagggcggcc ccctgccctt cgcctgggac atcctgtccc
     3481 cccagttcat gtacggctcc aaggcgtacg tgaagcaccc cgccgacatc cccgattaca
     3541 agaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg
     3601 gtctggtgac cgtgacccag gactcctccc tgcaggacgg cacgctgatc tacaaggtga
     3661 agatgcgcgg caccaacttc ccccccgacg gccccgtaat gcagaagaag accatgggct
     3721 gggaggcctc caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc gagatccacc
     3781 aggccctgaa gctgaaggac ggcggccgct acctggtgga gttcaagacc atctacatgg
     3841 ccaagaagcc cgtgcaactg cccggctact actacgtgga caccaagctg gacatcacct
     3901 cccacaacga ggactacacc atcgtggaac agtacgagcg ctccgagggc cgccaccacc
     3961 tgttcctgta cggcatggac gagctgtaca agtaagtcga ccttaaccct agaaagatag
     4021 tctgcgtaaa attgacgcat gcattcttga aatattgctc tctctttcta aatagcgcga
     4081 atccgtcgct gtgcatttag gacatctcag tcgccgcttg gagctcccgt gaggcgtgct
     4141 tgtcaatgcg gtaagtgtca ctgattttga actataacga ccgcgtgagt caaaatgacg
     4201 catgattatc ttttacgtga cttttaagat ttaactcata cgataattat attgttattt
     4261 catgttctac ttacgtgata acttattata tatatatttt cttgttatag atatcaggga
     4321 cagccatcat caactttgta tagaaaagtt gggctccggt gcccgtcagt gggcagagcg
     4381 cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa ccggtgccta
     4441 gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc
     4501 cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa
     4561 cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc ctggcctctt
     4621 tacgggttat ggcccttgcg tgccttgaat tacttccacc tggctgcagt acgtgattct
     4681 tgatcccgag cttcgggttg gaagtgggtg ggagagttcg aggccttgcg cttaaggagc
     4741 cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc gcgtgcgaat
     4801 ctggtggcac cttcgcgcct gtctcgctgc tttcgataag tctctagcca tttaaaattt
     4861 ttgatgacct gctgcgacgc tttttttctg gcaagatagt cttgtaaatg cgggccaaga
     4921 tctgcacact ggtatttcgg tttttggggc cgcgggcggc gacggggccc gtgcgtccca
     4981 gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg gacgggggta
     5041 gtctcaagct ggccggcctg ctctggtgcc tggtctcgcg ccgccgtgta tcgccccgcc
     5101 ctgggcggca aggctggccc ggtcggcacc agttgcgtga gcggaaagat ggccgcttcc
     5161 cggccctgct gcagggagct caaaatggag gacgcggcgc tcgggagagc gggcgggtga
     5221 gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat gtgactccac
     5281 ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg agcttttgga gtacgtcgtc
     5341 tttaggttgg ggggaggggt tttatgcgat ggagtttccc cacactgagt gggtggagac
     5401 tgaagttagg ccagcttggc acttgatgta attctccttg gaatttgccc tttttgagtt
     5461 tggatcttgg ttcattctca agcctcagac agtggttcaa agtttttttc ttccatttca
     5521 ggtgtcgtga aagcttacca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga
     5581 cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg
     5641 ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct
     5701 cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc
     5761 ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg
     5821 catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc
     5881 gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca
     5941 ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc
     6001 cggggtgccc gccttcctgg agacctccgc gccccacaac ctccccttct acgagcggct
     6061 cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac
     6121 ccgcaagccc ggtgccgcta gcgccactaa cttctccctg ttgaaacaag caggggatgt
     6181 cgaagagaat cccgggccaa tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     6241 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     6301 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     6361 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     6421 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     6481 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     6541 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     6601 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     6661 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     6721 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     6781 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     6841 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     6901 ggacgagctg tacaagtaac
//
