LOCUS       sequence-420244-        8682 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBac vector to express FLAG-tNGFR with zeocin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8682)
  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 8682)
  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_FLAG-tNGFR_ZeoR
FEATURES             Location/Qualifiers
     source          1..8682
                     /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..2675
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3420..3476
                     /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             3477..3851
                     /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    3867..4455
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3920..3940)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4338..4349)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4505..4626
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4542..4561)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4596..4615
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5389..5423
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5914..5930)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5914..5930)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5927..5949)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5938..5954
                     /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(5962..5968,5969..5986,5987..5992))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6007..6028
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6145..6162)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6316..6904)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6396..6415)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7075..7866,7867..7935))
                     /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     7698..7717
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7936..8040)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(8066..8521)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8203..8224)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8415..8434
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8648..8670
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8662..8679
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8663..8679
                     /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 ggactacaaa gacgatgacg acaagggagg aggccccaca ggcctgtaca
     2701 cacacagcgg tgagtgctgc aaagcctgca acctgggcga gggtgtggcc cagccttgtg
     2761 gagccaacca gaccgtgtgt gagccctgcc tggacagcgt gacgttctcc gacgtggtga
     2821 gcgcgaccga gccgtgcaag ccgtgcaccg agtgcgtggg gctccagagc atgtcggcgc
     2881 cgtgcgtgga ggccgacgac gccgtgtgcc gctgcgccta cggctactac caggatgaga
     2941 cgactgggcg ctgcgaggcg tgccgcgtgt gcgaggcggg ctcgggcctc gtgttctcct
     3001 gccaggacaa gcagaacacc gtgtgcgagg agtgccccga cggcacgtat tccgacgagg
     3061 ccaaccacgt ggacccgtgc ctgccctgca ccgtgtgcga ggacaccgag cgccagctcc
     3121 gcgagtgcac acgctgggcc gacgccgagt gcgaggagat ccctggccgt tggattacac
     3181 ggtccacacc cccagagggc tcggacagca cagcccccag cacccaggag cctgaggcac
     3241 ctccagaaca agacctcata gccagcacgg tggcaggtgt ggtgaccaca gtgatgggca
     3301 gctcccagcc cgtggtgacc cgaggcacca ccgacaacct catccctgtc tattgctcca
     3361 tcctggctgc tgtggttgtg ggccttgtgg cctacatagc cttcaagagg ggatccggcg
     3421 caacaaactt ctctctgctc aaacaagccg gcgacgtcga agagaatcct gggccgatgg
     3481 ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt
     3541 tctggaccga ccggctcggg ttctcccggg acttcgtgga ggacgacttc gccggtgtgg
     3601 tccgggacga cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg ccggacaaca
     3661 ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg
     3721 tgtccacgaa cttccgggac gcctccgggc cggccatgac cgagatcggc gagcagccgt
     3781 gggggcggga gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg
     3841 agcaggactg aacgcgttaa gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat
     3901 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     3961 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
     4021 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca
     4081 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt
     4141 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg
     4201 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga
     4261 aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt
     4321 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc
     4381 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt
     4441 gggccgcctc cccgcctcga gtctagaggg cccgtttaaa cccgctgatc agcctaacta
     4501 actaaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     4561 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     4621 atcttatcat gtctggaatt gactcaaatg atgtcaatta gtctatcaga agctcatctg
     4681 gtctcccttc cgggggacaa gacatccctg tttaatattt aaacagcagt gttcccaaac
     4741 tgggttctta tatcccttgc tctggtcaac caggttgcag ggtttcctgt cctcacagga
     4801 acgaagtccc taaagaaaca gtggcagcca ggtttagccc cggaattgac tggattcctt
     4861 ttttagggcc cattggtatg gctttttccc cgtatccccc caggtgtctg caggctcaaa
     4921 gagcagcgag aagcgttcag aggaaagcga tcccgtgcca ccttccccgt gcccgggctg
     4981 tccccgcacg ctgccggctc ggggatgcgg ggggagcgcc ggaccggagc ggagccccgg
     5041 gcggctcgct gctgccccct agcgggggag ggacgtaatt acatccctgg gggctttggg
     5101 ggggggctgt ccctgatatc tataacaaga aaatatatat ataataagtt atcacgtaag
     5161 tagaacatga aataacaata taattatcgt atgagttaaa tcttaaaagt cacgtaaaag
     5221 ataatcatgc gtcattttga ctcacgcggt cgttatagtt caaaatcagt gacacttacc
     5281 gcattgacaa gcacgcctca cgggagctcc aagcggcgac tgagatgtcc taaatgcaca
     5341 gcgacggatt cgcgctattt agaaagagag agcaatattt caagaatgca tgcgtcaatt
     5401 ttacgcagac tatctttcta gggttaatct agctgcatca ggatcatatc gtcgggtctt
     5461 ttttccggct cagtcatcgc ccaagctggc gctatctggg catcggggag gaagaagccc
     5521 gtgccttttc ccgcgaggtt gaagcggcat ggaaagagtt tgccgaggat gactgctgct
     5581 gcattgacgt tgagcgaaaa cgcacgttta ccatgatgat tcgggaaggt gtggccatgc
     5641 acgcctttaa cggtgaactg ttcgttcagg ccacctggga taccagttcg tcgcggcttt
     5701 tccggacaca gttccggatg gtcagcccga agcgcatcag caacccgaac aataccggcg
     5761 acagccggaa ctgccgtgcc ggtgtgcaga ttaatgacag cggtgcggcg ctgggatatt
     5821 acgtcagcga ggacgggtat cctggctgga tgccgcagaa atggacatgg ataccccgtg
     5881 agttacccgg cgggcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     5941 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     6001 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     6061 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     6121 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     6181 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     6241 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     6301 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     6361 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     6421 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     6481 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     6541 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     6601 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     6661 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     6721 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     6781 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     6841 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     6901 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     6961 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     7021 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     7081 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     7141 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     7201 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     7261 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     7321 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     7381 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     7441 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     7501 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     7561 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     7621 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     7681 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     7741 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     7801 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     7861 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     7921 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     7981 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     8041 cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg
     8101 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     8161 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     8221 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     8281 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     8341 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     8401 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     8461 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     8521 tcccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
     8581 ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca
     8641 gggttttccc agtcacgacg ttgtaaaacg acggccagtg ag
//
