LOCUS       pAMP345_(PB-CT3G-CERP2-MG-mNeonGreen)        8073 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-mCherry (constitutive rtTA and Puro resistance with CMV
            enhancer).
ACCESSION   urn.local...jm-czw1a8r
VERSION     urn.local...jm-czw1a8r
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:.:kv-czw1a8w"
                     /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:.:kw-czw1a8w"
                     /label="GBlock- AttB-GG Left (concatenated sequence 2)"
                     /note="Geneious type: Concatenated sequence"
     misc_feature    <365..366
                     /Source="referenceToDocument: urn:local:.:kx-czw1a8w"
                     /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..2162
                     /Source="referenceToDocument: urn:local:.:7k-czvzncv"
                     /label="IRES2-mNeonGreen (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..2156
                     /created_by="wyssuser"
                     /label="mNeonGreen"
     motif           2157..>2162
                     /created_by="wyssuser"
                     /label="NotI Clone Site"
     ligation        2159..2162
                     /label="Ligation"
     misc_feature    2159..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    <2159..2164
                     /Original_Bases
                     /label
                     /note="Geneious type: Editing History Insertion"
     ligation        2165..2182
                     /label="Ligation"
     polyA_signal    2174..2308
                     /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     2174..2199
                     /#_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(2282..2308)
                     /#_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        2309..2338
                     /label="Ligation"
     repeat_region   2580..2614
                     /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(2625..2642)
                     /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(2625..2641)
                     /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(2634..2656)
                     /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(2850..2869)
                     /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    2964..2971
                     /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     2971..2993
                     /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    3020..3026
                     /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(3032..3050)
                     /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        3118..3222
                     /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             3223..4083
                     /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(3441..3460)
                     /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    3938..3943
                     /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    3939
                     /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    3942
                     /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      4254..4842
                     /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     4743..4762
                     /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     4996..5013
                     /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    5022
                     /Original_Bases="A"
                     /label="A"
                     /note="Geneious type: Editing History Replacement"
     misc_feature    5034..5122
                     /Original_Bases
                     /label
                     /note="Geneious type: Editing History Insertion"
     protein_bind    5219..5240
                     /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        5255..5285
                     /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    5293..5309
                     /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     5298..5320
                     /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     5317..5333
                     /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     5317..5333
                     /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   5359..5421
                     /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    5597..5678
                     /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(5795..6394)
                     /created_by="wyssuser"
                     /label="Puromycin"
     misc_feature    5843
                     /Original_Bases="T"
                     /label="T"
                     /note="Geneious type: Editing History Replacement"
     ligation        <6395..6408
                     /label="Ligation"
     CDS             complement(6395..6451)
                     /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(6452..7204)
                     /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(7205..7228)
                     /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      7228..7237
                     /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(7257..7468)
                     /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(7469..7848)
                     /created_by="wyssuser"
                     /modified_by="wyssuser"
                     /label="CMV Enhancer"
     misc_feature    7481
                     /Original_Bases="G"
                     /label="G"
                     /note="Geneious type: Editing History Replacement"
     misc_feature    7519
                     /Original_Bases="C"
                     /label="C"
                     /note="Geneious type: Editing History Replacement"
     misc_feature    7670
                     /Original_Bases="C"
                     /label="C"
                     /note="Geneious type: Editing History Replacement"
     misc_feature    7849..8040
                     /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 caaccatggt cagcaaaggc gaagaagata acatggcatc actgccagcg acacacgaac
     1501 ttcatatttt cggctccatc aatggtgtgg acttcgatat ggttggacag ggtactggga
     1561 accctaacga tggatatgag gagttgaact tgaaaagtac caagggcgat cttcagtttt
     1621 caccttggat tctcgtacct catataggtt atggctttca tcaatatctg ccctatcctg
     1681 acggtatgtc acctttccaa gcagctatgg ttgacgggag cggttatcag gttcaccgga
     1741 caatgcagtt tgaagatgga gcctctttga ccgtaaatta tcggtatact tatgagggca
     1801 gccatattaa aggcgaggct caagttaagg gaactgggtt ccccgcggat ggtcctgtaa
     1861 tgacaaacag tctgacggct gcagactggt gccgctctaa gaaaacatat ccaaatgaca
     1921 agacgattat tagcactttc aagtggtcat acactacagg aaatggtaaa cgctatagga
     1981 gcactgcccg gaccacgtat actttcgcga agccgatggc ggcaaattat ttgaaaaacc
     2041 aaccgatgta tgtgttcaga aagacagaac tcaagcactc taaaactgaa ctgaacttca
     2101 aagagtggca gaaagccttt actgatgtaa tgggcatgga cgaactctat aagtaagcgg
     2161 ccgctttctc cgcgatccag acatgataag atacattgat gagtttggac aaaccaaaac
     2221 tagaatgcag tgaaaaaaat gccttatttg tgaaatttgt gatgctattg ccttatttgt
     2281 aaccattata agctgcaata aacaagttat ctataacaag aaaatatata tataataagt
     2341 tatcacgtaa gtagaacatg aaataacaat ataattatcg tatgagttaa atcttaaaag
     2401 tcacgtaaaa gataatcatg cgtcattttg actcacgcgg tcgttatagt tcaaaatcag
     2461 tgacacttac cgcattgaca agcacgcctc acgggagctc caagcggcga ctgagatgtc
     2521 ctaaatgcac agcgacggat tcgcgctatt tagaaagaga gagcaatatt tcaagaatgc
     2581 atgcgtcaat tttacgcaga ctatctttct agggttaaga attcactggc cgtcgtttta
     2641 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     2701 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     2761 cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     2821 atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     2881 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     2941 tccgcttaca gacaagctgt gaccatgtct ccgggagctg catgtgtcag aggttttcac
     3001 cgtcatcacc gaaacacgcg agttcgaaag ggcctcgtga tacgcctatt tttataggtt
     3061 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     3121 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     3181 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     3241 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     3301 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     3361 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     3421 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     3481 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     3541 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     3601 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     3661 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     3721 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     3781 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     3841 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     3901 tggtttattg ctgataaatc tggagccggt gagcgtggtt cacgcggtat cattgcagca
     3961 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     4021 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     4081 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     4141 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     4201 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     4261 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     4321 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     4381 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     4441 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     4501 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     4561 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     4621 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     4681 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     4741 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     4801 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     4861 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     4921 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     4981 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg atgagcgccc aatacgcaaa
     5041 ccgcctctca ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     5101 tggaaagcgg gcagtgagcg caacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     5161 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     5221 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
     5281 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
     5341 acgccaaggt cgacttaacc ctagaaagat aatcatattg tgacgtacgt taaagataat
     5401 catgcgtaaa attgacgcat gtgttttatc ggtctgtata tcgaggttta tttattaatt
     5461 tgaatagata ttaagtttta ttatatttac acttacatac taataataaa ttcaacaaac
     5521 aatttattta tgtttattta tttattaaaa aaaaacaaaa actcaaaatt tcttctataa
     5581 agtaacaaaa cttttagcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
     5641 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
     5701 ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca
     5761 aatgtggtat ggctgattat gatcctctgg agattcatgc gccaggtttt ctggtcatgc
     5821 accacgttcg tgggccctca ggaacctcca cgtcagcggt aactgtgaag cccagtcgct
     5881 cgtaaaaagg aagattccgg ggagcactgg tttcgaggaa cgccggcacg ccagcccgtt
     5941 ccgcggcctc aacaccagga agaacgacag cgcttcccaa acctttacct tggtgatctg
     6001 ggctgacacc gacagtggca agaaaccacg caggctcctt tggtctatga ggagccagca
     6061 agccctccat ttgctgttgt gccgccaacc tactccctga cagctcggcc atccgcggac
     6121 ctatttccgc gaacactgcg ccggcttcga cgctttccgg agtagtccag accgcaactg
     6181 cggcaccatc gtccgcaacc caaactttac caatgtccag acccacgcgc gtaagaaaca
     6241 gctcttgcaa ctcagttacc ctctctatgt gtctgtccgg atctacggta tggcgggtcg
     6301 ctggataatc tgcaaatgct gcggcaagtg ttcggacagc ccgtggtaca tcgtcacggg
     6361 tagcgagcct aacagtgggc ttatattccg tcatggggcc ggggttctcc tccacgtcgc
     6421 cggcctgctt cagcaggctg aagttggtgg cgccgctgcc cccggggagc atgtcaaggt
     6481 caaaatcgtc aagagcgtca gcaggcagca tatcaaggtc aaagtcgtca agggcatcgg
     6541 ctgggagcat gtctaagtca aaatcgtcaa gggcgtcggt cggcccgccg ctttcgcact
     6601 ttagctgttt ctccaggcca catatgatta gttccaggcc gaaaaggaag gcaggttcgg
     6661 ctccctgccg gtcgaacagc tcaattgctt gtttcagaag tgggggcata gaatcggtgg
     6721 taggtgtctc tctttcctct tttgctactt gatgctcctg ttcctccaat acgcagccca
     6781 gtgtaaagtg gcccacggcg gacagagcgt acagtgcgtt ctccagggag aagccttgct
     6841 gacacaggaa cgcgagctga ttttccaggg tttcgtactg tttctctgtt gggcgggtgc
     6901 cgagatgcac tttagccccg tcacgatgtg agaggagagc acagcggtat gacttggcgt
     6961 tgttccgcag aaagtcttgc catgactcgc cttccagggg gcaggagtgg gtatgatgcc
     7021 tgtccagcat ctcgattggc agggcatcga gcagggcccg cttgttcttc acgtgccagt
     7081 acagggtagg ctgctcaact cccagctttt gagcgagttt ccttgtcgtc aggccttcga
     7141 taccgactcc attgagtaat tccagagcag agtttatgac tttgctcttg tccagtctag
     7201 acatcttatc gtcatcgtct ttgtaatcca tggtggcgga tcccgcgtca cgacacctgt
     7261 gttctggcgg caaacccgtt gcgaaaaaga acgttcacgg cgactactgc acttatatac
     7321 ggttctcccc caccctcggg aaaaaggcgg agccagtaca cgacatcact ttcccagttt
     7381 accccgcgcc accttctcta ggcaccggtt caattgccga cccctccccc caacttctcg
     7441 ggcactgtgg gccatgtgcg ctctgcccca tggtaatagc catgactaat acgtagatgt
     7501 actgccaagt aggaaagtgc cataaggtca tgtactgggc ataatgccag gcgggccatt
     7561 taccgtcatt gacgtcaata gggggcgtac ttggcatatg atacacttga tgtactgcca
     7621 agtgggcagt ttaccgtaaa tactccaccc attgacgtca atggaaagtg cctattggcg
     7681 ttactatggg aacatacgtc attattgacg tcaatgggcg ggggtcgttg ggcggtcagc
     7741 caggcgggcc atttaccgta agttatgtaa cgcggaactc catatatggg ctatgaacta
     7801 atgaccccgt aattgattac tattaataac tagtcaataa tcaatgtcat aagctctaat
     7861 cgcataatcg agctaagagc gattaatatg cttagctagc ttagctaagc tagatttctc
     7921 tattctccca caatatatag cagctattag cttagcagtc cgaatacgat cgtccacgat
     7981 ccataagcgt aacacatttg agtagcgact gacgggcacc ggagccactc gagtggaatt
     8041 actgacgggc accggagcca ctcgagtgga att
//
