LOCUS       sequence-217664-        5766 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBac Transposon vector with a Gateway destination cassette 
            (neoPheS selection).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5766)
  AUTHORS   Chong ZS, Ohnishi S, Yusa K, Wright GJ
  TITLE     Pooled extracellular receptor-ligand interaction screening using 
            CRISPR activation.
  JOURNAL   Genome Biol. 2018 Nov 26;19(1):205. doi: 10.1186/s13059-018-1581-3.
  PUBMED    30477585
REFERENCE   2  (bases 1 to 5766)
  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: "Genome 
            Biol."; date: "2018-11-26"; volume: "19(1):205. doi"; pages: " 
            10.1186/s13059-018-1581-3"
COMMENT     Sequence Label: pPB-R1R2-EM7neoPheS
FEATURES             Location/Qualifiers
     source          1..5766
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(413..537)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     repeat_region   719..781
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(832..852)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(832..850)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(871..887)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(871..887)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(884..906)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    895..911
                     /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(919..925,926..943,944..949))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    964..985
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1102..1119)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1273..1861)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1353..1372)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2032..2823,2824..2892))
                     /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     2655..2674
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2893..2997)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3023..3478)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3160..3181)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3372..3391
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3605..3627
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3619..3636
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3620..3636
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3646..3665
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3646..3664
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   3694..3728
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    4018..4142
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        4161..4208
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4227..5030
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4290..4309)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4900..4919
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 ttcctgcgta acttctttga ggaagatttg cagattcgct tccgtccttc ctacttcccg
       61 tttaccgaac cttctgcaga agtggacgtc atgggtaaaa acggtaaatg gctggaagtg
      121 ctgggctgcg ggatggtgca tccgaacgtg ttgcgtaacg ttggcatcga cccggaagtt
      181 tactctggtt tcggcttcgg gatggggatg gagcgtctga ctatgttgcg ttacggcgtc
      241 accgacctgc gttcattctt cgaaaacgat ctgcgtttcc tcaaacagtt taaataaggc
      301 aggaatagat tatgaaattc agtgaactgt ggttacgcga atgggtgaac ccggcgattg
      361 atagcgatgc gctggcaaat caaatcacta tggcgctcta gtcgagtcta gacatagtga
      421 ctggatatgt tgtgttttac agtattatgt agtctgtttt ttatgcaaaa tctaatttaa
      481 tatattgata tttatatcat tttacgtttc tcgttcagct ttcttgtaca aagtggtact
      541 agttaaaagt tttgttactt tatagaagaa attttgagtt tttgtttttt tttaataaat
      601 aaataaacat aaataaattg tttgttgaat ttattattag tatgtaagtg taaatataat
      661 aaaacttaat atctattcaa attaataaat aaacctcgat atacagaccg ataaaacaca
      721 tgcgtcaatt ttacgcatga ttatctttaa cgtacgtcac aatatgatta tctttctagg
      781 gttaatctag tatacgcgta tgcggccgct taattaatcc agcttttgtt ccctttagtg
      841 agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
      901 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
      961 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     1021 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     1081 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     1141 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     1201 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     1261 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     1321 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     1381 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     1441 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     1501 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     1561 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     1621 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     1681 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     1741 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     1801 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     1861 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     1921 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     1981 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     2041 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     2101 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     2161 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     2221 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     2281 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     2341 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     2401 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     2461 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     2521 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     2581 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     2641 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     2701 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     2761 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     2821 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     2881 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     2941 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     3001 atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta
     3061 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     3121 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     3181 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     3241 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     3301 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     3361 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     3421 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc
     3481 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     3541 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     3601 ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgcgtaata cgactcacta
     3661 tagggcgaat tggggcgcgc cattctagat taaccctaga aagatagtct gcgtaaaatt
     3721 gacgcatgca ttcttgaaat attgctctct ctttctaaat agcgcgaatc cgtcgctgtg
     3781 catttaggac atctcagtcg ccgcttggag ctcccgtgag gcgtgcttgt caatgcggta
     3841 agtgtcactg attttgaact ataacgaccg cgtgagtcaa aatgacgcat gattatcttt
     3901 tacgtgactt ttaagattta actcatacga taattatatt gttatttcat gttctactta
     3961 cgtgataact tattatatat atattttctt gttatagata tcaactagaa tgctagcaca
     4021 agtttgtaca aaaaagctga acgagaaacg taaaatgata taaatatcaa tatattaaat
     4081 tagattttgc ataaaaaaca gactacataa tactgtaaaa cacaacatat ccagtcacta
     4141 tgggatccga attcaagctt gttgacaatt aatcatcggc atagtatatc ggcatagtat
     4201 aatacgacaa ggtgaggaac taaaccatgg gatcggccat tgaacaagat ggattgcacg
     4261 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     4321 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     4381 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt
     4441 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     4501 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     4561 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     4621 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     4681 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     4741 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg
     4801 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     4861 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     4921 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     4981 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga ctcgaaattc
     5041 gaacgaacca gtgtcaccac tgacacaatg aggaaaacca tgtcacatct cgcagaactg
     5101 gttgccagtg cgaaggcggc cattagccag gcgtcagatg ttgccgcgtt agataatgtg
     5161 cgcgtcgaat atttgggtaa aaaagggcac ttaacccttc agatgacgac cctgcgtgag
     5221 ctgccgccag aagagcgtcc ggcagctggt gcggttatca acgaagcgaa agagcaggtt
     5281 cagcaggcgc tgaatgcgcg taaagcggaa ctggaaagcg ctgcactgaa tgcgcgtctg
     5341 gcggcggaaa cgattgatgt ctctctgcca ggtcgtcgca ttgaaaacgg cggtctgcat
     5401 ccggttaccc gtaccatcga ccgtatcgaa agtttcttcg gtgagcttgg ctttaccgtg
     5461 gcaaccgggc cggaaatcga agacgattat cataacttcg atgctctgaa cattcctggt
     5521 caccacccgg cgcgcgctga ccacgacact ttctggtttg acactacccg cctgctgcgt
     5581 acccagacct ctggcgtaca gatccgcacc atgaaagccc agcagccacc gattcgtatc
     5641 atcgcgcctg gccgtgttta tcgtaacgac tacgaccaga ctcacacgcc gatgttccat
     5701 cagatggaag gtctgattgt tgataccaac atcagcttta ccaacctgaa aggcacgctg
     5761 cacgac
//
