LOCUS       sequence-420249-        8640 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBac vector to express tNGFR with zeocin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8640)
  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 8640)
  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_tNGFR_ZeoR
FEATURES             Location/Qualifiers
     source          1..8640
                     /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             3378..3434
                     /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             3435..3809
                     /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    3825..4413
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3878..3898)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4296..4307)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4463..4584
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4500..4519)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4554..4573
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5347..5381
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5872..5888)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5872..5888)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5885..5907)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5896..5912
                     /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(5920..5926,5927..5944,5945..5950))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5965..5986
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6103..6120)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6274..6862)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6354..6373)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7033..7824,7825..7893))
                     /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     7656..7675
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7894..7998)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(8024..8479)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8161..8182)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8373..8392
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8606..8628
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8620..8637
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8621..8637
                     /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 gccccacagg cctgtacaca cacagcggtg agtgctgcaa agcctgcaac ctgggcgagg
     2701 gtgtggccca gccttgtgga gccaaccaga ccgtgtgtga gccctgcctg gacagcgtga
     2761 cgttctccga cgtggtgagc gcgaccgagc cgtgcaagcc gtgcaccgag tgcgtggggc
     2821 tccagagcat gtcggcgccg tgcgtggagg ccgacgacgc cgtgtgccgc tgcgcctacg
     2881 gctactacca ggatgagacg actgggcgct gcgaggcgtg ccgcgtgtgc gaggcgggct
     2941 cgggcctcgt gttctcctgc caggacaagc agaacaccgt gtgcgaggag tgccccgacg
     3001 gcacgtattc cgacgaggcc aaccacgtgg acccgtgcct gccctgcacc gtgtgcgagg
     3061 acaccgagcg ccagctccgc gagtgcacac gctgggccga cgccgagtgc gaggagatcc
     3121 ctggccgttg gattacacgg tccacacccc cagagggctc ggacagcaca gcccccagca
     3181 cccaggagcc tgaggcacct ccagaacaag acctcatagc cagcacggtg gcaggtgtgg
     3241 tgaccacagt gatgggcagc tcccagcccg tggtgacccg aggcaccacc gacaacctca
     3301 tccctgtcta ttgctccatc ctggctgctg tggttgtggg ccttgtggcc tacatagcct
     3361 tcaagagggg atccggcgca acaaacttct ctctgctcaa acaagccggc gacgtcgaag
     3421 agaatcctgg gccgatggcc aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg
     3481 tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg
     3541 acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg gtccaggacc
     3601 aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg cggcctggac gagctgtacg
     3661 ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc ctccgggccg gccatgaccg
     3721 agatcggcga gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg
     3781 tgcacttcgt ggccgaggag caggactgaa cgcgttaagt cgacaatcaa cctctggatt
     3841 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     3901 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     3961 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
     4021 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
     4081 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
     4141 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
     4201 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct
     4261 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
     4321 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
     4381 cgagtcggat ctccctttgg gccgcctccc cgcctcgagt ctagagggcc cgtttaaacc
     4441 cgctgatcag cctaactaac taaacttgtt tattgcagct tataatggtt acaaataaag
     4501 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     4561 gtccaaactc atcaatgtat cttatcatgt ctggaattga ctcaaatgat gtcaattagt
     4621 ctatcagaag ctcatctggt ctcccttccg ggggacaaga catccctgtt taatatttaa
     4681 acagcagtgt tcccaaactg ggttcttata tcccttgctc tggtcaacca ggttgcaggg
     4741 tttcctgtcc tcacaggaac gaagtcccta aagaaacagt ggcagccagg tttagccccg
     4801 gaattgactg gattcctttt ttagggccca ttggtatggc tttttccccg tatcccccca
     4861 ggtgtctgca ggctcaaaga gcagcgagaa gcgttcagag gaaagcgatc ccgtgccacc
     4921 ttccccgtgc ccgggctgtc cccgcacgct gccggctcgg ggatgcgggg ggagcgccgg
     4981 accggagcgg agccccgggc ggctcgctgc tgccccctag cgggggaggg acgtaattac
     5041 atccctgggg gctttggggg ggggctgtcc ctgatatcta taacaagaaa atatatatat
     5101 aataagttat cacgtaagta gaacatgaaa taacaatata attatcgtat gagttaaatc
     5161 ttaaaagtca cgtaaaagat aatcatgcgt cattttgact cacgcggtcg ttatagttca
     5221 aaatcagtga cacttaccgc attgacaagc acgcctcacg ggagctccaa gcggcgactg
     5281 agatgtccta aatgcacagc gacggattcg cgctatttag aaagagagag caatatttca
     5341 agaatgcatg cgtcaatttt acgcagacta tctttctagg gttaatctag ctgcatcagg
     5401 atcatatcgt cgggtctttt ttccggctca gtcatcgccc aagctggcgc tatctgggca
     5461 tcggggagga agaagcccgt gccttttccc gcgaggttga agcggcatgg aaagagtttg
     5521 ccgaggatga ctgctgctgc attgacgttg agcgaaaacg cacgtttacc atgatgattc
     5581 gggaaggtgt ggccatgcac gcctttaacg gtgaactgtt cgttcaggcc acctgggata
     5641 ccagttcgtc gcggcttttc cggacacagt tccggatggt cagcccgaag cgcatcagca
     5701 acccgaacaa taccggcgac agccggaact gccgtgccgg tgtgcagatt aatgacagcg
     5761 gtgcggcgct gggatattac gtcagcgagg acgggtatcc tggctggatg ccgcagaaat
     5821 ggacatggat accccgtgag ttacccggcg ggcgcgcttg gcgtaatcat ggtcatagct
     5881 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     5941 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     6001 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     6061 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     6121 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     6181 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     6241 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     6301 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     6361 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     6421 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     6481 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     6541 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     6601 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     6661 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     6721 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     6781 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     6841 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     6901 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     6961 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     7021 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     7081 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     7141 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     7201 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     7261 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     7321 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     7381 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     7441 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     7501 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     7561 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     7621 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     7681 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     7741 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     7801 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     7861 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     7921 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     7981 acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat
     8041 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     8101 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     8161 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     8221 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     8281 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     8341 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     8401 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     8461 gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     8521 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
     8581 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag
//
