LOCUS       sequence-420265-        8910 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBac vector to express HA-tNGFR with puromycin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8910)
  AUTHORS   Najia MA, Borrajo J, Le A, Tsai F, Huang JY, Griffith LG, Daley GQ, 
            Blainey PC
  TITLE     Live-cell transcriptomics with engineered virus-like particles
  JOURNAL   bioRxiv 2024.10.01.616098
REFERENCE   2  (bases 1 to 8910)
  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: "bioRxiv 
            2024.10.01.616098"
COMMENT     Sequence Label: pPB_EF1a_HA-tNGFR_PuroR
FEATURES             Location/Qualifiers
     source          1..8910
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   127..189
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1356..2534
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1587..2525
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2482..2502
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      2541..2550
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2652..2678
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3423..3479
                     /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             3480..4079
                     /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(3480..3499)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3976..3996
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    4095..4683
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4148..4168)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4566..4577)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4733..4854
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4770..4789)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4824..4843
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5617..5651
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6142..6158)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6142..6158)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6155..6177)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6166..6182
                     /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(6190..6196,6197..6214,6215..6220))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6235..6256
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6373..6390)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6544..7132)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6624..6643)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7303..8094,8095..8163))
                     /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     7926..7945
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8164..8268)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(8294..8749)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8431..8452)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8643..8662
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8876..8898
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8890..8907
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8891..8907
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 cgcgcctcgt tcattcacgt ttttgaaccc gtggaggacg ggcagactcg cggtgcaaat
       61 gtgttttaca gcgtgatgga gcagatgaag atgctcgaca cgctgcagaa cacgcagcta
      121 gattaaccct agaaagataa tcatattgtg acgtacgtta aagataatca tgtgtaaaat
      181 tgacgcatgt gttttatcgg tctgtatatc gaggtttatt tattaatttg aatagatatt
      241 aagttttatt atatttacac ttacatacta ataataaatt caacaaacaa tttatttatg
      301 tttatttatt tattaaaaaa aacaaaaact caaaatttct tctataaagt aacaaaactt
      361 ttatgaggga cagccccccc ccaaagcccc cagggatgta attacgtccc tcccccgcta
      421 gggggcagca gcgagccgcc cggggctccg ctccggtccg gcgctccccc cgcatccccg
      481 agccggcagc gtgcggggac agcccgggca cggggaaggt ggcacgggat cgctttcctc
      541 tgaacgcttc tcgctgctct ttgagcctgc agacacctgg ggggatacgg ggaaaaggcc
      601 tccaccataa agatggttaa ttaacccacc caagatctgg atcgataagc ttgatatcga
      661 attcgggagg tggtccctgc agttacgcca atgataaccc ccgccagaaa aatcttagta
      721 gccttccctt tttgttttcc gtgccccaac tcggcggatt gactcggccc cttccggaaa
      781 cacccgaatc aacttctagt caaattattg ttcacgccgc aatgacccac ccctggcccg
      841 cgtctgtgga actgacccct ggtgtacagg agagttcgct gctgaaagtg gtcccaaagg
      901 ggtactagtt tttaagctcc caactccccc tcccccagcg tctggaggat tccacaccct
      961 cgcaccgcag gggcgaggaa gtgggcggag tccggttttg gcgccagccg ctgaggctgc
     1021 caagcagaaa agccaccgct gaggagactc cggtcactgt cctcgccccg cctccccctt
     1081 ccctcccctt ggggaccacc gggcgccacg ccgcgaacgg taagtgccgc ggtcgtcggc
     1141 gcctccgccc tccccctagg gccccaattc ccagcgggcg cggcgcgcgg ccccgcgcgc
     1201 agctcccggc tccctccccc ttcggatgtg gcttgagctg taggcgcgga gtcctgcagc
     1261 cccgataaaa taaaagattt tatttagtct ccagaaaaag gggggaatga aagaccccac
     1321 ctgtaggttt ggcaatcttg aaaggagtgg gaattggctc cggtgcccgt cagtgggcag
     1381 agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgatccggtg
     1441 cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt
     1501 ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc
     1561 gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc
     1621 tctttacggg ttatggccct tgcgtgcctt gaattacttc cacctggctg cagtacgtga
     1681 ttcttgatcc cgagcttcgg gttggaagtg ggtgggagag ttcgaggcct tgcgcttaag
     1741 gagccccttc gcctcgtgct tgagttgagg cctggcctgg gcgctggggc cgccgcgtgc
     1801 gaatctggtg gcaccttcgc gcctgtctcg ctgctttcga taagtctcta gccatttaaa
     1861 atttttgatg acctgctgcg acgctttttt tctggcaaga tagtcttgta aatgcgggcc
     1921 aagatctgca cactggtatt tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt
     1981 cccagcgcac atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga atcggacggg
     2041 ggtagtctca agctggccgg cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc
     2101 cgccctgggc ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa agatggccgc
     2161 ttcccggccc tgctgcaggg agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg
     2221 gtgagtcacc cacacaaagg aaaagggcct ttccgtcctc agccgtcgct tcatgtgact
     2281 ccacggagta ccgggcgccg tccaggcacc tcgattagtt ctcgagcttt tggagtacgt
     2341 cgtctttagg ttggggggag gggttttatg cgatggagtt tccccacact gagtgggtgg
     2401 agactgaagt taggccagct tggcacttga tgtaattctc cttggaattt gccctttttg
     2461 agtttggatc ttggttcatt ctcaagcctc agacagtggt tcaaagtttt tttcttccat
     2521 ttcaggtgtc gtgacgtacg gccaccatgg gggcaggtgc caccggccgc gccatggacg
     2581 ggccgcgcct gctgctgttg ctgcttctgg gggtgtccct tggaggtgcc aaggaggcat
     2641 gcgggggagg gtacccatac gatgttccag attacgctgg aggaggcccc acaggcctgt
     2701 acacacacag cggtgagtgc tgcaaagcct gcaacctggg cgagggtgtg gcccagcctt
     2761 gtggagccaa ccagaccgtg tgtgagccct gcctggacag cgtgacgttc tccgacgtgg
     2821 tgagcgcgac cgagccgtgc aagccgtgca ccgagtgcgt ggggctccag agcatgtcgg
     2881 cgccgtgcgt ggaggccgac gacgccgtgt gccactgcgc ctacggctac taccaggatg
     2941 agacgactgg gcgctgcgag gcgtgccgcg tgtgcgaggc gggctcgggc ctcgtgttct
     3001 cctgccagga caagcagaac accgtgtgcg aggagtgccc cgacggcacg tattccgacg
     3061 aggccaacca cgtggacccg tgcctgccct gcaccgtgtg cgaggacacc gagcgccagc
     3121 tccgcgagtg cacacgctgg gccgacgccg agtgcgagga gatccctggc cgttggatta
     3181 cacggtccac acccccagag ggctcggaca gcacagcccc cagcacccag gagcctgagg
     3241 cacctccaga acaagacctc atagccagca cggtggcagg tgtggtgacc acagtgatgg
     3301 gcagctccca gcccgtggtg acccgaggca ccaccgacaa cctcatccct gtctattgct
     3361 ccatcctggc tgctgtggtt gtgggccttg tggcctacat agccttcaag aggggatccg
     3421 gcgcaacaaa cttctctctg ctcaaacaag ccggcgacgt cgaagagaat cctgggccga
     3481 tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac
     3541 gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc
     3601 gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca
     3661 tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga
     3721 gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt
     3781 cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc
     3841 ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag ggtctgggca
     3901 gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg
     3961 agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg
     4021 acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctaaa
     4081 cgcgttaagt cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
     4141 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
     4201 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc
     4261 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg
     4321 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg
     4381 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga
     4441 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct
     4501 ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg
     4561 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc
     4621 ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc
     4681 cgcctcgagt ctagagggcc cgtttaaacc cgctgatcag cctaactaac taaacttgtt
     4741 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     4801 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     4861 ctggaattga ctcaaatgat gtcaattagt ctatcagaag ctcatctggt ctcccttccg
     4921 ggggacaaga catccctgtt taatatttaa acagcagtgt tcccaaactg ggttcttata
     4981 tcccttgctc tggtcaacca ggttgcaggg tttcctgtcc tcacaggaac gaagtcccta
     5041 aagaaacagt ggcagccagg tttagccccg gaattgactg gattcctttt ttagggccca
     5101 ttggtatggc tttttccccg tatcccccca ggtgtctgca ggctcaaaga gcagcgagaa
     5161 gcgttcagag gaaagcgatc ccgtgccacc ttccccgtgc ccgggctgtc cccgcacgct
     5221 gccggctcgg ggatgcgggg ggagcgccgg accggagcgg agccccgggc ggctcgctgc
     5281 tgccccctag cgggggaggg acgtaattac atccctgggg gctttggggg ggggctgtcc
     5341 ctgatatcta taacaagaaa atatatatat aataagttat cacgtaagta gaacatgaaa
     5401 taacaatata attatcgtat gagttaaatc ttaaaagtca cgtaaaagat aatcatgcgt
     5461 cattttgact cacgcggtcg ttatagttca aaatcagtga cacttaccgc attgacaagc
     5521 acgcctcacg ggagctccaa gcggcgactg agatgtccta aatgcacagc gacggattcg
     5581 cgctatttag aaagagagag caatatttca agaatgcatg cgtcaatttt acgcagacta
     5641 tctttctagg gttaatctag ctgcatcagg atcatatcgt cgggtctttt ttccggctca
     5701 gtcatcgccc aagctggcgc tatctgggca tcggggagga agaagcccgt gccttttccc
     5761 gcgaggttga agcggcatgg aaagagtttg ccgaggatga ctgctgctgc attgacgttg
     5821 agcgaaaacg cacgtttacc atgatgattc gggaaggtgt ggccatgcac gcctttaacg
     5881 gtgaactgtt cgttcaggcc acctgggata ccagttcgtc gcggcttttc cggacacagt
     5941 tccggatggt cagcccgaag cgcatcagca acccgaacaa taccggcgac agccggaact
     6001 gccgtgccgg tgtgcagatt aatgacagcg gtgcggcgct gggatattac gtcagcgagg
     6061 acgggtatcc tggctggatg ccgcagaaat ggacatggat accccgtgag ttacccggcg
     6121 ggcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     6181 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     6241 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     6301 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     6361 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     6421 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     6481 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     6541 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     6601 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     6661 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     6721 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     6781 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     6841 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     6901 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     6961 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     7021 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     7081 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     7141 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     7201 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     7261 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     7321 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     7381 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     7441 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     7501 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     7561 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     7621 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     7681 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     7741 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     7801 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     7861 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     7921 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     7981 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     8041 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     8101 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     8161 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     8221 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     8281 aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     8341 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     8401 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     8461 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     8521 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     8581 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     8641 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     8701 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
     8761 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
     8821 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
     8881 tcacgacgtt gtaaaacgac ggccagtgag
//
