LOCUS       sequence-420248-        8685 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBac vector to express HA-tNGFR with zeocin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8685)
  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 8685)
  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_ZeoR
FEATURES             Location/Qualifiers
     source          1..8685
                     /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..3854
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     misc_feature    3870..4458
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3923..3943)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4341..4352)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4508..4629
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4545..4564)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4599..4618
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5392..5426
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5917..5933)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5917..5933)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5930..5952)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5941..5957
                     /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(5965..5971,5972..5989,5990..5995))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6010..6031
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6148..6165)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6319..6907)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6399..6418)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7078..7869,7870..7938))
                     /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     7701..7720
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7939..8043)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(8069..8524)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8206..8227)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8418..8437
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8651..8673
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8665..8682
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8666..8682
                     /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 gccgctgcgc 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 tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg
     3541 agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg
     3601 tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca
     3661 acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg
     3721 tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc
     3781 cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg
     3841 aggagcagga ctgaacgcgt taagtcgaca atcaacctct ggattacaaa atttgtgaaa
     3901 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     3961 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     4021 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     4081 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     4141 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     4201 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     4261 ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     4321 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     4381 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     4441 tttgggccgc ctccccgcct cgagtctaga gggcccgttt aaacccgctg atcagcctaa
     4501 ctaactaaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     4561 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     4621 tgtatcttat catgtctgga attgactcaa atgatgtcaa ttagtctatc agaagctcat
     4681 ctggtctccc ttccggggga caagacatcc ctgtttaata tttaaacagc agtgttccca
     4741 aactgggttc ttatatccct tgctctggtc aaccaggttg cagggtttcc tgtcctcaca
     4801 ggaacgaagt ccctaaagaa acagtggcag ccaggtttag ccccggaatt gactggattc
     4861 cttttttagg gcccattggt atggcttttt ccccgtatcc ccccaggtgt ctgcaggctc
     4921 aaagagcagc gagaagcgtt cagaggaaag cgatcccgtg ccaccttccc cgtgcccggg
     4981 ctgtccccgc acgctgccgg ctcggggatg cggggggagc gccggaccgg agcggagccc
     5041 cgggcggctc gctgctgccc cctagcgggg gagggacgta attacatccc tgggggcttt
     5101 gggggggggc tgtccctgat atctataaca agaaaatata tatataataa gttatcacgt
     5161 aagtagaaca tgaaataaca atataattat cgtatgagtt aaatcttaaa agtcacgtaa
     5221 aagataatca tgcgtcattt tgactcacgc ggtcgttata gttcaaaatc agtgacactt
     5281 accgcattga caagcacgcc tcacgggagc tccaagcggc gactgagatg tcctaaatgc
     5341 acagcgacgg attcgcgcta tttagaaaga gagagcaata tttcaagaat gcatgcgtca
     5401 attttacgca gactatcttt ctagggttaa tctagctgca tcaggatcat atcgtcgggt
     5461 cttttttccg gctcagtcat cgcccaagct ggcgctatct gggcatcggg gaggaagaag
     5521 cccgtgcctt ttcccgcgag gttgaagcgg catggaaaga gtttgccgag gatgactgct
     5581 gctgcattga cgttgagcga aaacgcacgt ttaccatgat gattcgggaa ggtgtggcca
     5641 tgcacgcctt taacggtgaa ctgttcgttc aggccacctg ggataccagt tcgtcgcggc
     5701 ttttccggac acagttccgg atggtcagcc cgaagcgcat cagcaacccg aacaataccg
     5761 gcgacagccg gaactgccgt gccggtgtgc agattaatga cagcggtgcg gcgctgggat
     5821 attacgtcag cgaggacggg tatcctggct ggatgccgca gaaatggaca tggatacccc
     5881 gtgagttacc cggcgggcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     5941 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     6001 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     6061 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     6121 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     6181 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     6241 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     6301 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     6361 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     6421 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     6481 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     6541 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     6601 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     6661 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     6721 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     6781 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     6841 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     6901 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     6961 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     7021 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     7081 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     7141 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     7201 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     7261 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     7321 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     7381 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     7441 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     7501 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     7561 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     7621 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     7681 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     7741 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     7801 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     7861 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     7921 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     7981 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     8041 gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc
     8101 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     8161 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     8221 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     8281 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     8341 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     8401 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     8461 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     8521 gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
     8581 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg
     8641 ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgag
//
