LOCUS       sequence-212988-        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"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   674..708
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    998..1122
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        1141..1188
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             1207..2010
                     /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(1270..1289)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1880..1899
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    complement(3159..3283)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     repeat_region   3465..3527
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3578..3598)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3578..3596)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3617..3633)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3617..3633)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3630..3652)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3641..3657
                     /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(3665..3671,3672..3689,3690..3695))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3710..3731
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3848..3865)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4019..4607)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4099..4118)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4778..5569,5570..5638))
                     /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     5401..5420
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5639..5743)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggggcgcgc
      661 cattctagat taaccctaga aagatagtct gcgtaaaatt gacgcatgca ttcttgaaat
      721 attgctctct ctttctaaat agcgcgaatc cgtcgctgtg catttaggac atctcagtcg
      781 ccgcttggag ctcccgtgag gcgtgcttgt caatgcggta agtgtcactg attttgaact
      841 ataacgaccg cgtgagtcaa aatgacgcat gattatcttt tacgtgactt ttaagattta
      901 actcatacga taattatatt gttatttcat gttctactta cgtgataact tattatatat
      961 atattttctt gttatagata tcaactagaa tgctagcaca agtttgtaca aaaaagctga
     1021 acgagaaacg taaaatgata taaatatcaa tatattaaat tagattttgc ataaaaaaca
     1081 gactacataa tactgtaaaa cacaacatat ccagtcacta tgggatccga attcaagctt
     1141 gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac
     1201 taaaccatgg gatcggccat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     1261 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     1321 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     1381 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     1441 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     1501 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     1561 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     1621 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     1681 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     1741 gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     1801 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     1861 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     1921 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     1981 ttctatcgcc ttcttgacga gttcttctga ctcgaaattc gaacgaacca gtgtcaccac
     2041 tgacacaatg aggaaaacca tgtcacatct cgcagaactg gttgccagtg cgaaggcggc
     2101 cattagccag gcgtcagatg ttgccgcgtt agataatgtg cgcgtcgaat atttgggtaa
     2161 aaaagggcac ttaacccttc agatgacgac cctgcgtgag ctgccgccag aagagcgtcc
     2221 ggcagctggt gcggttatca acgaagcgaa agagcaggtt cagcaggcgc tgaatgcgcg
     2281 taaagcggaa ctggaaagcg ctgcactgaa tgcgcgtctg gcggcggaaa cgattgatgt
     2341 ctctctgcca ggtcgtcgca ttgaaaacgg cggtctgcat ccggttaccc gtaccatcga
     2401 ccgtatcgaa agtttcttcg gtgagcttgg ctttaccgtg gcaaccgggc cggaaatcga
     2461 agacgattat cataacttcg atgctctgaa cattcctggt caccacccgg cgcgcgctga
     2521 ccacgacact ttctggtttg acactacccg cctgctgcgt acccagacct ctggcgtaca
     2581 gatccgcacc atgaaagccc agcagccacc gattcgtatc atcgcgcctg gccgtgttta
     2641 tcgtaacgac tacgaccaga ctcacacgcc gatgttccat cagatggaag gtctgattgt
     2701 tgataccaac atcagcttta ccaacctgaa aggcacgctg cacgacttcc tgcgtaactt
     2761 ctttgaggaa gatttgcaga ttcgcttccg tccttcctac ttcccgttta ccgaaccttc
     2821 tgcagaagtg gacgtcatgg gtaaaaacgg taaatggctg gaagtgctgg gctgcgggat
     2881 ggtgcatccg aacgtgttgc gtaacgttgg catcgacccg gaagtttact ctggtttcgg
     2941 cttcgggatg gggatggagc gtctgactat gttgcgttac ggcgtcaccg acctgcgttc
     3001 attcttcgaa aacgatctgc gtttcctcaa acagtttaaa taaggcagga atagattatg
     3061 aaattcagtg aactgtggtt acgcgaatgg gtgaacccgg cgattgatag cgatgcgctg
     3121 gcaaatcaaa tcactatggc gctctagtcg agtctagaca tagtgactgg atatgttgtg
     3181 ttttacagta ttatgtagtc tgttttttat gcaaaatcta atttaatata ttgatattta
     3241 tatcatttta cgtttctcgt tcagctttct tgtacaaagt ggtactagtt aaaagttttg
     3301 ttactttata gaagaaattt tgagtttttg ttttttttta ataaataaat aaacataaat
     3361 aaattgtttg ttgaatttat tattagtatg taagtgtaaa tataataaaa cttaatatct
     3421 attcaaatta ataaataaac ctcgatatac agaccgataa aacacatgcg tcaattttac
     3481 gcatgattat ctttaacgta cgtcacaata tgattatctt tctagggtta atctagtata
     3541 cgcgtatgcg gccgcttaat taatccagct tttgttccct ttagtgaggg ttaattgcgc
     3601 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     3661 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     3721 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     3781 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     3841 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     3901 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     3961 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4021 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     4081 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4141 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4201 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4261 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4321 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4381 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     4441 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4501 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4561 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     4621 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4681 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     4741 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     4801 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     4861 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     4921 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     4981 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     5041 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     5101 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     5161 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     5221 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     5281 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     5341 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     5401 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     5461 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     5521 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     5581 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     5641 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     5701 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     5761 tgccac
//
