LOCUS       pAMP343_(PB-CT3G-CERP2-MG-IRES-mTagBFP2)        8064 bp    DNA     circular UNA 13-FEB-2021
DEFINITION  All-in-One piggyBac transposon MG vector for dox-inducible (CMV
            minimal promoter-T3G) expression of a polycistronic cassette of an
            ORF -IRES-mTagBFP2 (constitutive rtTA and Puro resistance with CMV
            enhancer).
ACCESSION   urn.local...jk-czw1a8q
VERSION     urn.local...jk-czw1a8q
KEYWORDS    .
SOURCE      
  ORGANISM  .
FEATURES             Location/Qualifiers
     promoter        9..>262
                     /note="3rd-generation Tet-responsive promoter that can be
                     activated by binding of Tet-On(R) 3G, modified to
                     eliminate binding sites for endogenous mammalian
                     transcription factors"
                     /locus_tag="TRE3GS promoter"
                     /ApEinfo_label="TRE3GS promoter"
                     /ApEinfo_fwdcolor="#346ee0"
                     /ApEinfo_revcolor="#346ee0"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="TRE3GS promoter"
     protein_bind    17..35
                     /gene="tetO"
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /locus_tag="tet operator"
                     /ApEinfo_label="tet operator"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="tet operator"
     protein_bind    53..71
                     /gene="tetO"
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /locus_tag="tet operator(1)"
                     /ApEinfo_label="tet operator"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="tet operator(1)"
     protein_bind    89..107
                     /gene="tetO"
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /locus_tag="tet operator(2)"
                     /ApEinfo_label="tet operator"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="tet operator(2)"
     protein_bind    125..143
                     /gene="tetO"
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /locus_tag="tet operator(3)"
                     /ApEinfo_label="tet operator"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="tet operator(3)"
     protein_bind    161..179
                     /gene="tetO"
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /locus_tag="tet operator(4)"
                     /ApEinfo_label="tet operator"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="tet operator(4)"
     protein_bind    197..215
                     /gene="tetO"
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /locus_tag="tet operator(5)"
                     /ApEinfo_label="tet operator"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="tet operator(5)"
     protein_bind    233..251
                     /gene="tetO"
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /locus_tag="tet operator(6)"
                     /ApEinfo_label="tet operator"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="tet operator(6)"
     ligation        259..262
                     /label="Ligation"
     source          <263..>264
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
                     /locus_tag="source:synthetic DNA construct(2)"
                     /ApEinfo_label="source:synthetic DNA construct"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="source:synthetic DNA construct(2)"
     promoter        265..364
                     /created_by="wyssuser"
                     /modified_by="Alex"
                     /label="mCMV Promoter"
     ligation        330..347
                     /label="Ligation"
     misc_feature    <365..>367
                     /Source="referenceToDocument: urn:local:.:kl-czw1a8t"
                     /label="Double Gate Vector-BsaI (digested by HindIII,
                     KpnI) Fragment 2 (concatenated sequence 1)"
                     /note="Geneious type: Concatenated sequence"
     misc_feature    <365..>367
                     /Source="referenceToDocument: urn:local:.:km-czw1a8t"
                     /label="GBlock- AttB-GG Left (concatenated sequence 2)"
                     /note="Geneious type: Concatenated sequence"
     misc_feature    <365..366
                     /Source="referenceToDocument: urn:local:.:kn-czw1a8t"
                     /label="mCMV-Tet-Insert (digested by HindIII, KpnI)
                     Fragment 2 (concatenated sequence 2)"
                     /note="Geneious type: Concatenated sequence"
     source          <365..>366
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
                     /locus_tag="source:synthetic DNA construct(2)"
                     /ApEinfo_label="source:synthetic DNA construct"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="source:synthetic DNA construct(2)"
     primer_bind     365..389
                     /%GC="36.0"
                     /Hairpin_Tm="39.9"
                     /Self_Dimer_Tm="22.1"
                     /Tm="59.4"
                     /Pair_Dimer_Tm="None"
                     /Sequence="ACCACAAGTTTGTACAAAAAAGCTG"
                     /created_by="primer3"
                     /Product_Size=390
                     /annotation_group="2049510240876995fb70dc8-7d7b-4d70-af46-
                     b8c33b2782a3cT3G-attR1_MG_GA_Left-clean"
                     /#_Off-target_Sites=0
                     /Off-target_Template="cT3G-attR1_MG_GA_Left-clean"
                     /Extension="GACCTCCATAGAAGACAC"
                     /label="cT3G-attR1_MG_GA_Left-clean (1 F) -
                     CK461-PB-cT3G-cERP2-Puro Base Vector #4-CMV_Enhancer -
                     mCMV-Tet-Promoter (digested by EcoRV, BbsI) Fragment 4"
     protein_bind    368..492
                     /bound_moiety="<html><body>LR Clonase™</body></html>"
                     /gene="<html><body>mutant version
                     of&nbsp;<i>att</i>R</body></html>"
                     /note="<html><body>recombination site for the Gateway® LR
                     reaction</body></html>"
                     /label="attR1"
     attR1           369..492
                     /label="attR1"
     motif           508..525
                     /created_by="wyssuser"
                     /label="I-SceI"
     CDS             526..570
                     /created_by="wyssuser"
                     /modified_by="wyssuser"
                     /label="Trademark"
     motif           complement(571..597)
                     /created_by="wyssuser"
                     /label="I-CeuI"
     primer_bind     complement(593..612)
                     /Pair_Dimer_Tm="9.2"
                     /Product_Size=266
                     /#_Off-target_Sites=0
                     /Off-target_Template="cT3G-ATTR1-MG-Left"
                     /Mismatches=0
                     /Extension="GTCACTATG"
                     /%GC="40.0"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Tm="53.6"
                     /annotation_group="750483074821766f5e104a-67a6-4667-80f2-e
                     b6f7ac51df3"
                     /Sequence="ATGGAGCTTTCCTTCTAACT"
                     /created_by="primer3"
                     /label="cT3G-ATTR1-MG-Left (266 R) - ATTR2-Right-FP
                     modular"
     misc_feature    598..612
                     /Original_Bases="CTAAGGATAGATTCAGTTGCTCAATTAGATAAGAAGAGTTA
                     GATAGCGAATAGGGATAACAGGGTAATAGGCTGGCTGCACATAGTGAC"
                     /label="CTAAGGATAGATTCAGTTGCTCAATTAGATAAGAAGAGTTAGATAGCGAA
                     TAGGGATAACAGGGTAATAGGCTGGCTGCACATAGTGAC"
                     /note="Geneious type: Editing History Replacement"
     ligation        604..621
                     /label="Ligation"
     protein_bind    complement(613..737)
                     /bound_moiety="<html><body>LR Clonase™</body></html>"
                     /gene="<html><body>mutant version of
                     <i>att</i>R</body></html>"
                     /note="<html><body>recombination site for the Gateway® LR
                     reaction</body></html>"
                     /label="attR2"
     primer_bind     613..635
                     /Pair_Dimer_Tm="None"
                     /Product_Size=274
                     /#_Off-target_Sites=0
                     /Off-target_Template="ATTR2-Right-FP modular"
                     /Mismatches=0
                     /Extension="AAGCTCCAT"
                     /%GC="39.1"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Tm="56.1"
                     /annotation_group="7509071802962029489636-5040-4bea-b51f-4
                     a5e13970535"
                     /Sequence="CATAGTGACTGGATATGTTGTGT"
                     /created_by="primer3"
                     /label="ATTR2-Right-FP modular (1 F) - cT3G-ATTR1-MG-Left"
     attR2           complement(613..736)
                     /label="attR2"
     misc_feature    738
                     /Original_Bases
                     /label
                     /note="Geneious type: Editing History Insertion"
     CDS             739..765
                     /created_by="wyssuser"
                     /label="HA Tag"
     terminator      766..774
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
                     /locus_tag="source:synthetic DNA construct(4)"
                     /ApEinfo_label="source:synthetic DNA construct"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /modified_by="wyssuser"
                     /label="STOPS"
     primer_bind     778..800
                     /Sequence="ATACTCAGAAGATGTCACTCACC"
                     /Hairpin_Tm="None"
                     /Tm="57.4"
                     /Self_Dimer_Tm="None"
                     /%GC="43.5"
                     /created_by="primer3"
                     /Extension="ACACTCTTTCCCTACACGACGCTCTTCCGATCTNN"
                     /modified_by="Alex"
                     /label="BC_Fwd"
     source          <806..>824
                     /created_by="wyssuser"
                     /label="BBSI Cloning"
     CDS             <806..>824
                     /created_by="wyssuser"
                     /codon_start=3
                     /label="BsaI Cloning"
     misc_feature    806..825
                     /Original_Bases="CACCGGGTCTTCGAGAAGACCTGTT"
                     /label="CACCGGGTCTTCGAGAAGACCTGTT"
                     /note="Geneious type: Editing History Replacement"
     primer_bind     complement(830..852)
                     /Sequence="CATTCATAGTTCTTGCTCAGTGG"
                     /Hairpin_Tm="None"
                     /Tm="57.5"
                     /Self_Dimer_Tm="None"
                     /%GC="43.5"
                     /created_by="primer3"
                     /Extension="GAGTTCAGACGTGTGCTCTTCCGATCTNNN"
                     /modified_by="Alex"
                     /label="BC_Rev"
     misc_feature    853..>857
                     /Original_Bases="GCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGA
                     GGAGAACCCCGGCCCC"
                     /label="GCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCC
                     CGGCCCC"
                     /note="Geneious type: Editing History Replacement"
     misc_feature    854..2153
                     /Source="referenceToDocument: urn:local:.:76-czvzlth"
                     /label="IRES2-mTagBFP2 (digested by BglII, NotI) Fragment
                     2 (concatenated sequence 2)"
                     /note="Geneious type: Concatenated sequence"
     motif           <854..858
                     /created_by="wyssuser"
                     /label="BgIII Clone Site"
     ligation        854..857
                     /label="Ligation"
     intron          859..1445
                     /created_by="wyssuser"
                     /label="IRES2"
     CDS             1446..2147
                     /created_by="wyssuser"
                     /label="mTAGBFP2_Human Optimized"
     motif           2148..>2153
                     /created_by="wyssuser"
                     /label="NotI Clone Site"
     ligation        2150..2153
                     /label="Ligation"
     misc_feature    2150..857
                     /Source="referenceToDocument: urn:local:.:6o-czvzjqi"
                     /label="pAMP247 (PB-CT3G-CERP2-MG) (digested by NotI,
                     BglII) Fragment 2 (concatenated sequence 1)"
                     /note="Geneious type: Concatenated sequence"
     misc_feature    <2150..2155
                     /Original_Bases
                     /label
                     /note="Geneious type: Editing History Insertion"
     ligation        2156..2173
                     /label="Ligation"
     polyA_signal    2165..2299
                     /note="SV40 polyadenylation signal"
                     /locus_tag="SV40 poly(A) signal"
                     /ApEinfo_label="SV40 poly(A) signal"
                     /ApEinfo_fwdcolor="#ff3eee"
                     /ApEinfo_revcolor="#ff3eee"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="SV40 poly(A) signal"
     primer_bind     2165..2190
                     /#_Off-target_Sites=0
                     /Off-target_Template="PolyA"
                     /Mismatches=0
                     /Extension="TTTCTCCGC"
                     /%GC="34.6"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Tm="56.7"
                     /annotation_group="74822804194994fd2e5350-3751-4d48-b167-7
                     808cb75bb1f"
                     /Sequence="GATCCAGACATGATAAGATACATTGA"
                     /created_by="primer3"
                     /label="PolyA (1 F) - ATTR2-Right-FP modular"
     primer_bind     complement(2273..2299)
                     /#_Off-target_Sites=0
                     /Off-target_Template="PolyA"
                     /Mismatches=0
                     /Extension="TTATTATATATATATTTTCTTGTTATAGAT"
                     /%GC="29.6"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Tm="58.5"
                     /annotation_group="74822854197151f1be6ca2-142e-499f-9aa1-e
                     154a3a53574"
                     /Sequence="AACTTGTTTATTGCAGCTTATAATGGT"
                     /created_by="primer3"
                     /label="PolyA (135 R) - CK461-PB-cT3G-cERP2-Puro Base
                     Vector #4-CMV_Enhancer - mCMV-Tet-Promoter (digested by
                     EcoRV, BbsI) Fragment 4"
     ligation        2300..2329
                     /label="Ligation"
     repeat_region   2571..2605
                     /note="piggyBac transposon-specific inverted terminal
                     repeat sequence (ITR)"
                     /locus_tag="piggyBac left (5') inverted repeat"
                     /ApEinfo_label="piggyBac left (5') inverted repeat"
                     /ApEinfo_fwdcolor="#0dfff7"
                     /ApEinfo_revcolor="#0dfff7"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="piggyBac left (5') inverted repeat"
     primer_bind     complement(2616..2633)
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21)
                     Forward"
                     /locus_tag="M13 Forward"
                     /ApEinfo_label="M13 Forward"
                     /ApEinfo_fwdcolor="#14c0bd"
                     /ApEinfo_revcolor="#4ec02b"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="M13 Forward"
     primer_bind     complement(2616..2632)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /locus_tag="M13 fwd"
                     /ApEinfo_label="M13 fwd"
                     /ApEinfo_fwdcolor="#14c0bd"
                     /ApEinfo_revcolor="#4ec02b"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="M13 fwd"
     primer_bind     complement(2625..2647)
                     /note="In lacZ gene"
                     /locus_tag="M13/pUC Forward"
                     /ApEinfo_label="M13/pUC Forward"
                     /ApEinfo_fwdcolor="#14c0bd"
                     /ApEinfo_revcolor="#4ec02b"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="M13/pUC Forward"
     primer_bind     complement(2841..2860)
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
                     /locus_tag="pRS-marker"
                     /ApEinfo_label="pRS-marker"
                     /ApEinfo_fwdcolor="#14c0bd"
                     /ApEinfo_revcolor="#4ec02b"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="pRS-marker"
     misc_feature    2955..2962
                     /locus_tag="bsmbi"
                     /ApEinfo_label="bsmbi"
                     /ApEinfo_fwdcolor="#fb0106"
                     /ApEinfo_revcolor="green"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="bsmbi"
     primer_bind     2962..2984
                     /note="pGEX vectors, reverse primer"
                     /locus_tag="pGEX 3'"
                     /ApEinfo_label="pGEX 3'"
                     /ApEinfo_fwdcolor="#14c0bd"
                     /ApEinfo_revcolor="#4ec02b"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="pGEX 3'"
     misc_feature    3011..3017
                     /locus_tag="bsmbi(1)"
                     /ApEinfo_label="bsmbi"
                     /ApEinfo_fwdcolor="#fb0106"
                     /ApEinfo_revcolor="green"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="bsmbi(1)"
     primer_bind     complement(3023..3041)
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
                     /locus_tag="pBRforEco"
                     /ApEinfo_label="pBRforEco"
                     /ApEinfo_fwdcolor="#14c0bd"
                     /ApEinfo_revcolor="#4ec02b"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="pBRforEco"
     promoter        3109..3213
                     /gene="bla"
                     /locus_tag="AmpR promoter"
                     /ApEinfo_label="AmpR promoter"
                     /ApEinfo_fwdcolor="#346ee0"
                     /ApEinfo_revcolor="#346ee0"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="AmpR promoter"
     CDS             3214..4074
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="confers resistance to ampicillin, carbenicillin,
                     and related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGAS LIKHW"
                     /locus_tag="AmpR"
                     /ApEinfo_label="AmpR"
                     /ApEinfo_fwdcolor="#e9d024"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="AmpR"
     primer_bind     complement(3432..3451)
                     /note="Ampicillin resistance gene, reverse primer"
                     /locus_tag="Amp-R"
                     /ApEinfo_label="Amp-R"
                     /ApEinfo_fwdcolor="#14c0bd"
                     /ApEinfo_revcolor="#4ec02b"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="Amp-R"
     misc_feature    3929..3934
                     /locus_tag="bsaI"
                     /ApEinfo_label="bsaI"
                     /ApEinfo_fwdcolor="#fb0106"
                     /ApEinfo_revcolor="green"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="bsaI"
     misc_feature    3930
                     /locus_tag="recoded"
                     /ApEinfo_label="recoded"
                     /ApEinfo_fwdcolor="#66ffcb"
                     /ApEinfo_revcolor="green"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="recoded"
     misc_feature    3933
                     /locus_tag="recoded(1)"
                     /ApEinfo_label="recoded"
                     /ApEinfo_fwdcolor="#6666fe"
                     /ApEinfo_revcolor="green"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="recoded(1)"
     rep_origin      4245..4833
                     /direction="RIGHT"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /locus_tag="ori"
                     /ApEinfo_label="ori"
                     /ApEinfo_fwdcolor="#999999"
                     /ApEinfo_revcolor="#999999"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="ori"
     primer_bind     4734..4753
                     /note="pBR322 origin, forward primer"
                     /locus_tag="pBR322ori-F"
                     /ApEinfo_label="pBR322ori-F"
                     /ApEinfo_fwdcolor="#14c0bd"
                     /ApEinfo_revcolor="#4ec02b"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="pBR322ori-F"
     primer_bind     4987..5004
                     /note="L4440 vector, forward primer"
                     /locus_tag="L4440"
                     /ApEinfo_label="L4440"
                     /ApEinfo_fwdcolor="#14c0bd"
                     /ApEinfo_revcolor="#4ec02b"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="L4440"
     misc_feature    5013
                     /Original_Bases="A"
                     /label="A"
                     /note="Geneious type: Editing History Replacement"
     misc_feature    5025..5113
                     /Original_Bases
                     /label
                     /note="Geneious type: Editing History Insertion"
     protein_bind    5210..5231
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the
                     presenceof cAMP."
                     /locus_tag="CAP binding site"
                     /ApEinfo_label="CAP binding site"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="CAP binding site"
     promoter        5246..5276
                     /note="promoter for the E. coli lac operon"
                     /locus_tag="lac promoter"
                     /ApEinfo_label="lac promoter"
                     /ApEinfo_fwdcolor="#346ee0"
                     /ApEinfo_revcolor="#346ee0"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="lac promoter"
     protein_bind    5284..5300
                     /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)."
                     /locus_tag="lac operator"
                     /ApEinfo_label="lac operator"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="lac operator"
     primer_bind     5289..5311
                     /note="In lacZ gene"
                     /locus_tag="M13/pUC Reverse"
                     /ApEinfo_label="M13/pUC Reverse"
                     /ApEinfo_fwdcolor="#14c0bd"
                     /ApEinfo_revcolor="#4ec02b"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="M13/pUC Reverse"
     primer_bind     5308..5324
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /locus_tag="M13 rev"
                     /ApEinfo_label="M13 rev"
                     /ApEinfo_fwdcolor="#14c0bd"
                     /ApEinfo_revcolor="#4ec02b"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="M13 rev"
     primer_bind     5308..5324
                     /note="In lacZ gene. Also called M13-rev"
                     /locus_tag="M13 Reverse"
                     /ApEinfo_label="M13 Reverse"
                     /ApEinfo_fwdcolor="#14c0bd"
                     /ApEinfo_revcolor="#4ec02b"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="M13 Reverse"
     repeat_region   5350..5412
                     /note="piggyBac transposon-specific inverted terminal
                     repeat sequence (ITR)"
                     /locus_tag="piggyBac right (3') inverted repeat"
                     /ApEinfo_label="piggyBac right (3') inverted repeat"
                     /ApEinfo_fwdcolor="#0dfff7"
                     /ApEinfo_revcolor="#0dfff7"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="piggyBac right (3') inverted repeat"
     polyA_signal    5588..5669
                     /note="SV40 polyadenylation signal"
                     /locus_tag="SV40 poly(A) signal(1)"
                     /ApEinfo_label="SV40 poly(A) signal"
                     /ApEinfo_fwdcolor="#ff3eee"
                     /ApEinfo_revcolor="#ff3eee"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="SV40 poly(A) signal(1)"
     CDS             complement(5786..6385)
                     /created_by="wyssuser"
                     /label="Puromycin"
     misc_feature    5834
                     /Original_Bases="T"
                     /label="T"
                     /note="Geneious type: Editing History Replacement"
     ligation        <6386..6399
                     /label="Ligation"
     CDS             complement(6386..6442)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1
                     polyprotein"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond
                     between the Gly and Pro residues, yielding separate
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
                     /locus_tag="P2A"
                     /ApEinfo_label="P2A"
                     /ApEinfo_fwdcolor="#e9d024"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="P2A"
     CDS             complement(6443..7195)
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to
                     promoters containing the tet operator in the presence of
                     doxycycline"
                     /locus_tag="Tet-On(R) 3G"
                     /ApEinfo_label="Tet-On(R) 3G"
                     /ApEinfo_fwdcolor="#e9d024"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /Original_Translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLG
                     VEQPTLYWHVKNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSH
                     RDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEH
                     QVAKEERETPTTDSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPT
                     DALDDFDLDMLPA DALDDFDLDMLPADALDDFDLDMLPG"
                     /modified_by="Alex"
                     /label="Tet-On(R) 3G"
     CDS             complement(7196..7219)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an
                     enterokinasecleavage site"
                     /translation="DYKDDDDK"
                     /locus_tag="FLAG"
                     /ApEinfo_label="FLAG"
                     /ApEinfo_fwdcolor="#e9d024"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="FLAG"
     regulatory      7219..7228
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong
                     initiationof translation (Kozak, 1987)"
                     /locus_tag="vertebrate consensus sequence for strong
                     initiation of translation (Kozak, 1987)"
                     /ApEinfo_label="vertebrate consensus sequence for strong
                     initiation of translation (Kozak, 1987)"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="vertebrate consensus sequence for strong
                     initiation of translation (Kozak, 1987)"
     promoter        complement(7248..7459)
                     /note="core promoter for human elongation factor
                     EF-1-alpha"
                     /locus_tag="EF-1-alpha core promoter"
                     /ApEinfo_label="EF-1-alpha core promoter"
                     /ApEinfo_fwdcolor="#346ee0"
                     /ApEinfo_revcolor="#346ee0"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
                     /label="EF-1-alpha core promoter"
     promoter        complement(7460..7839)
                     /created_by="wyssuser"
                     /modified_by="wyssuser"
                     /label="CMV Enhancer"
     misc_feature    7472
                     /Original_Bases="G"
                     /label="G"
                     /note="Geneious type: Editing History Replacement"
     misc_feature    7510
                     /Original_Bases="C"
                     /label="C"
                     /note="Geneious type: Editing History Replacement"
     misc_feature    7661
                     /Original_Bases="C"
                     /label="C"
                     /note="Geneious type: Editing History Replacement"
     misc_feature    7840..8031
                     /Original_Bases="TTCAATTGCCGACCCCTCCCCCCAACTTCTCGGGGACTGTG
                     GGCGATGTGCGCTCTGCCC"
                     /label="TTCAATTGCCGACCCCTCCCCCCAACTTCTCGGGGACTGTGGGCGATGTG
                     CGCTCTGCCC"
                     /note="Geneious type: Editing History Replacement"
ORIGIN      
        1 atcacctcga gtttactccc tatcagtgat agagaacgta tgaagagttt actccctatc
       61 agtgatagag aacgtatgca gactttactc cctatcagtg atagagaacg tataaggagt
      121 ttactcccta tcagtgatag agaacgtatg accagtttac tccctatcag tgatagagaa
      181 cgtatctaca gtttactccc tatcagtgat agagaacgta tatccagttt actccctatc
      241 agtgatagag aacgtataag cttgcctata taagcagagc tcgtttagtg aaccgtcaga
      301 tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca
      361 gcctaccaca agtttgtaca aaaaagctga acgagaaacg taaaatgata taaatatcaa
      421 tatattaaat tagattttgc ataaaaaaca gactacataa tactgtaaaa cacaacatat
      481 ccagtcacta tggcgacaga agaagtatag ggataacagg gtaattgttg taagcgcgct
      541 atgatggagg ctatgccact agaatctgcg ttcgctacct taggaccgtt atagttagaa
      601 ggaaagctcc atcatagtga ctggatatgt tgtgttttac agtattatgt agtctgtttt
      661 ttatgcaaaa tctaatttaa tatattgata tttatatcat tttacgtttc tcgttcagct
      721 ttcttgtaca aagtggtata cccatacgat gttccagatt acgcttagta atgagaaata
      781 ctcagaagat gtcactcacc atagagagac cattaatggg tctcctaggc cactgagcaa
      841 gaactatgaa tgagatctcc cctctccctc ccccccccct aacgttactg gccgaagccg
      901 cttggaataa ggccggtgtg cgtttgtcta tatgttattt tccaccatat tgccgtcttt
      961 tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct
     1021 ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct
     1081 ggaagcttct tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaacccccc
     1141 acctggcgac aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaaggc
     1201 ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctctc
     1261 ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc
     1321 tgatctgggg cctcggtaca catgctttac atgtgtttag tcgaggttaa aaaaacgtct
     1381 aggccccccg aaccacgggg acgtggtttt cctttgaaaa acacgatgat aatatggcca
     1441 caaccatgag tgaactcata aaggaaaata tgcacatgaa actttatatg gaaggtacag
     1501 tcgacaatca ccatttcaaa tgtacctcag aaggagaggg caagccatac gaaggaaccc
     1561 aaaccatgcg aataaaggta gtggaggggg gtcctctccc ctttgcattt gatatcctcg
     1621 caacgtcttt cctgtatggc tcaaaaacct tcataaatca tacgcagggc ataccagact
     1681 tcttcaaaca gtcctttccg gagggcttca cttgggaaag agtcacgact tatgaagatg
     1741 gcggcgtcct gacagcaact caggacacat ctctacagga tggctgcctc atctataatg
     1801 tgaagattcg gggagttaac ttcactagca acggacctgt tatgcagaag aaaacgcttg
     1861 gttgggaagc atttacggaa actctttatc ctgctgatgg tgggcttgaa ggtcgcaacg
     1921 acatggcact caaactggtt ggagggagcc accttatcgc caatgcaaag acgacttatc
     1981 gctctaaaaa accagctaaa aacctgaaga tgcccggtgt gtactatgta gactaccgcc
     2041 tggaacgaat aaaagaagcc aataacgaga cttatgtgga gcagcatgaa gtggcggtgg
     2101 cacggtactg tgatctgccg agcaaacttg ggcataaact gaattaagcg gccgctttct
     2161 ccgcgatcca gacatgataa gatacattga tgagtttgga caaaccaaaa ctagaatgca
     2221 gtgaaaaaaa tgccttattt gtgaaatttg tgatgctatt gccttatttg taaccattat
     2281 aagctgcaat aaacaagtta tctataacaa gaaaatatat atataataag ttatcacgta
     2341 agtagaacat gaaataacaa tataattatc gtatgagtta aatcttaaaa gtcacgtaaa
     2401 agataatcat gcgtcatttt gactcacgcg gtcgttatag ttcaaaatca gtgacactta
     2461 ccgcattgac aagcacgcct cacgggagct ccaagcggcg actgagatgt cctaaatgca
     2521 cagcgacgga ttcgcgctat ttagaaagag agagcaatat ttcaagaatg catgcgtcaa
     2581 ttttacgcag actatctttc tagggttaag aattcactgg ccgtcgtttt acaacgtcgt
     2641 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     2701 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     2761 aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     2821 cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     2881 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     2941 agacaagctg tgaccatgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     3001 cgaaacacgc gagttcgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     3061 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     3121 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     3181 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     3241 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     3301 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     3361 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     3421 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     3481 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     3541 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     3601 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     3661 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     3721 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     3781 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     3841 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     3901 gctgataaat ctggagccgg tgagcgtggt tcacgcggta tcattgcagc actggggcca
     3961 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     4021 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     4081 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     4141 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     4201 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     4261 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     4321 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     4381 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     4441 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     4501 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     4561 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     4621 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     4681 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     4741 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     4801 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     4861 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     4921 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     4981 gagcgcagcg agtcagtgag cgaggaagcg gatgagcgcc caatacgcaa accgcctctc
     5041 accgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     5101 ggcagtgagc gcaacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     5161 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     5221 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     5281 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagg
     5341 tcgacttaac cctagaaaga taatcatatt gtgacgtacg ttaaagataa tcatgcgtaa
     5401 aattgacgca tgtgttttat cggtctgtat atcgaggttt atttattaat ttgaatagat
     5461 attaagtttt attatattta cacttacata ctaataataa attcaacaaa caatttattt
     5521 atgtttattt atttattaaa aaaaaacaaa aactcaaaat ttcttctata aagtaacaaa
     5581 acttttagca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     5641 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc
     5701 aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta
     5761 tggctgatta tgatcctctg gagattcatg cgccaggttt tctggtcatg caccacgttc
     5821 gtgggccctc aggaacctcc acgtcagcgg taactgtgaa gcccagtcgc tcgtaaaaag
     5881 gaagattccg gggagcactg gtttcgagga acgccggcac gccagcccgt tccgcggcct
     5941 caacaccagg aagaacgaca gcgcttccca aacctttacc ttggtgatct gggctgacac
     6001 cgacagtggc aagaaaccac gcaggctcct ttggtctatg aggagccagc aagccctcca
     6061 tttgctgttg tgccgccaac ctactccctg acagctcggc catccgcgga cctatttccg
     6121 cgaacactgc gccggcttcg acgctttccg gagtagtcca gaccgcaact gcggcaccat
     6181 cgtccgcaac ccaaacttta ccaatgtcca gacccacgcg cgtaagaaac agctcttgca
     6241 actcagttac cctctctatg tgtctgtccg gatctacggt atggcgggtc gctggataat
     6301 ctgcaaatgc tgcggcaagt gttcggacag cccgtggtac atcgtcacgg gtagcgagcc
     6361 taacagtggg cttatattcc gtcatggggc cggggttctc ctccacgtcg ccggcctgct
     6421 tcagcaggct gaagttggtg gcgccgctgc ccccggggag catgtcaagg tcaaaatcgt
     6481 caagagcgtc agcaggcagc atatcaaggt caaagtcgtc aagggcatcg gctgggagca
     6541 tgtctaagtc aaaatcgtca agggcgtcgg tcggcccgcc gctttcgcac tttagctgtt
     6601 tctccaggcc acatatgatt agttccaggc cgaaaaggaa ggcaggttcg gctccctgcc
     6661 ggtcgaacag ctcaattgct tgtttcagaa gtgggggcat agaatcggtg gtaggtgtct
     6721 ctctttcctc ttttgctact tgatgctcct gttcctccaa tacgcagccc agtgtaaagt
     6781 ggcccacggc ggacagagcg tacagtgcgt tctccaggga gaagccttgc tgacacagga
     6841 acgcgagctg attttccagg gtttcgtact gtttctctgt tgggcgggtg ccgagatgca
     6901 ctttagcccc gtcacgatgt gagaggagag cacagcggta tgacttggcg ttgttccgca
     6961 gaaagtcttg ccatgactcg ccttccaggg ggcaggagtg ggtatgatgc ctgtccagca
     7021 tctcgattgg cagggcatcg agcagggccc gcttgttctt cacgtgccag tacagggtag
     7081 gctgctcaac tcccagcttt tgagcgagtt tccttgtcgt caggccttcg ataccgactc
     7141 cattgagtaa ttccagagca gagtttatga ctttgctctt gtccagtcta gacatcttat
     7201 cgtcatcgtc tttgtaatcc atggtggcgg atcccgcgtc acgacacctg tgttctggcg
     7261 gcaaacccgt tgcgaaaaag aacgttcacg gcgactactg cacttatata cggttctccc
     7321 ccaccctcgg gaaaaaggcg gagccagtac acgacatcac tttcccagtt taccccgcgc
     7381 caccttctct aggcaccggt tcaattgccg acccctcccc ccaacttctc gggcactgtg
     7441 ggccatgtgc gctctgcccc atggtaatag ccatgactaa tacgtagatg tactgccaag
     7501 taggaaagtg ccataaggtc atgtactggg cataatgcca ggcgggccat ttaccgtcat
     7561 tgacgtcaat agggggcgta cttggcatat gatacacttg atgtactgcc aagtgggcag
     7621 tttaccgtaa atactccacc cattgacgtc aatggaaagt gcctattggc gttactatgg
     7681 gaacatacgt cattattgac gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc
     7741 catttaccgt aagttatgta acgcggaact ccatatatgg gctatgaact aatgaccccg
     7801 taattgatta ctattaataa ctagtcaata atcaatgtca taagctctaa tcgcataatc
     7861 gagctaagag cgattaatat gcttagctag cttagctaag ctagatttct ctattctccc
     7921 acaatatata gcagctatta gcttagcagt ccgaatacga tcgtccacga tccataagcg
     7981 taacacattt gagtagcgac tgacgggcac cggagccact cgagtggaat tactgacggg
     8041 caccggagcc actcgagtgg aatt
//
