LOCUS       pAMP438_(PB-CT3G-ERP2-MG)        6232 bp    DNA     circular UNA 01-APR-2021
DEFINITION  Gibson/Homology Cloning ligation product.
ACCESSION   urn.local...s-d0pj6dg
VERSION     urn.local...s-d0pj6dg
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"
                     /label="mCMV Promoter"
     misc_feature    265..364
                     /note="mCMV promoter"
                     /X-Transferred_From="Exported"
                     /X-Transferred_Similarity="100.00%"
                     /Transferred_From="PB-TA-ERN"
                     /Transferred_Similarity="100.00%"
                     /Transferred_From="CK270-pB-Tet-BFP-Puro, CK271-
                     pB-Tet-mCherry-Puro, CK272- pB-Tet-EYFP-Puro, CK273 -
                     pB-Tet-sfGFP-Puro"
                     /Transferred_Similarity="100.00%"
                     /label="mCMV promoter"
     misc            368..385
                     /Color="-1"
                     /label="Spacer"
     ligation        368..385
                     /label="Ligation"
     source          <381..>385
                     /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)"
     ligation        382..385
                     /label="Ligation"
     primer_bind     386..411
                     /Sequence="ACAAGTTTGTACAAAAAAGCTGAACG"
                     /Hairpin_Tm="40.2"
                     /Tm="59.9"
                     /Self_Dimer_Tm="22.1"
                     /%GC="34.6"
                     /created_by="primer3"
                     /Extension="CGGTGGCGGCATCGGTAC"
                     /modified_by="Alex"
                     /label="CK490_L"
     attR1           387..510
                     /label="attR1"
     motif           526..543
                     /created_by="wyssuser"
                     /label="I-SceI"
     CDS             544..588
                     /created_by="wyssuser"
                     /modified_by="Alex"
                     /label="Trademark"
     ligation        549..572
                     /label="Ligation"
     primer_bind     549..568
                     /#_Off-target_Sites=0
                     /Off-target_Template="Final Plasmid"
                     /modified_by="wyssuser"
                     /Mismatches=0
                     /%GC="55.0"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="36.7"
                     /Tm="60.0"
                     /annotation_group="70878786263964666171c1e-31ec-49b0-b427-
                     ff07b800632d"
                     /Sequence="TAAGCGCGCTATGATGGAGG"
                     /created_by="primer3"
                     /label="Trademark_Fwd"
     primer_bind     complement(553..572)
                     /#_Off-target_Sites=0
                     /Off-target_Template="Final Plasmid"
                     /modified_by="wyssuser"
                     /Mismatches=0
                     /%GC="55.0"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Tm="59.8"
                     /annotation_group="708787907780060fd0790b6-6856-46c6-bb75-
                     b3713c4bf7d8"
                     /Sequence="ATAGCCTCCATCATAGCGCG"
                     /created_by="primer3"
                     /label="Trademark_Rev"
     motif           complement(589..615)
                     /created_by="wyssuser"
                     /label="I-CeuI"
     primer_bind     complement(611..630)
                     /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    616..630
                     /Original_Bases="CTAAGGATAGATTCAGTTGCTCAATTAGATAAGAAGAGTTA
                     GATAGCGAATAGGGATAACAGGGTAATAGGCTGGCTGCACATAGTGAC"
                     /label="CTAAGGATAGATTCAGTTGCTCAATTAGATAAGAAGAGTTAGATAGCGAA
                     TAGGGATAACAGGGTAATAGGCTGGCTGCACATAGTGAC"
                     /note="Geneious type: Editing History Replacement"
     ligation        622..639
                     /label="Ligation"
     attR2           complement(631..754)
                     /label="attR2"
     primer_bind     631..653
                     /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"
     misc_feature    756
                     /Original_Bases
                     /label
                     /note="Geneious type: Editing History Insertion"
     CDS             757..783
                     /created_by="wyssuser"
                     /label="HA Tag"
     terminator      784..792
                     /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     796..818
                     /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          <824..>842
                     /created_by="wyssuser"
                     /label="BBSI Cloning"
     misc_feature    824..843
                     /Original_Bases="CACCGGGTCTTCGAGAAGACCTGTT"
                     /label="CACCGGGTCTTCGAGAAGACCTGTT"
                     /note="Geneious type: Editing History Replacement"
     CDS             <824..>842
                     /created_by="wyssuser"
                     /codon_start=3
                     /label="BsaI Cloning"
     primer_bind     complement(848..870)
                     /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    871..876
                     /Original_Bases="GCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGA
                     GGAGAACCCCGGCCCC"
                     /label="GCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCC
                     CGGCCCC"
                     /note="Geneious type: Editing History Replacement"
     misc_feature    879..885
                     /Original_Bases
                     /label
                     /note="Geneious type: Editing History Insertion"
     misc_feature    888..895
                     /Original_Bases
                     /label
                     /note="Geneious type: Editing History Insertion"
     primer_bind     complement(889..904)
                     /Pair_Dimer_Tm="None"
                     /Product_Size=274
                     /#_Off-target_Sites=0
                     /Off-target_Template="ATTR2-Right-FP modular"
                     /Mismatches=0
                     /Extension="GTCTGGATC"
                     /%GC="75.0"
                     /Hairpin_Tm="47.3"
                     /Self_Dimer_Tm="24.1"
                     /Tm="61.3"
                     /annotation_group="75090758895449787a8e9e-e407-4646-bf1b-f
                     1ef6b1429e2"
                     /Sequence="GCGGAGAAAGCGGCCG"
                     /created_by="primer3"
                     /label="ATTR2-Right-FP modular (274 R) - PolyA"
     ligation        896..913
                     /label="Ligation"
     polyA_signal    905..1039
                     /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     905..930
                     /#_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(1013..1039)
                     /#_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"
     primer_bind     complement(1014..1039)
                     /Sequence="AACTTGTTTATTGCAGCTTATAATGG"
                     /Hairpin_Tm="None"
                     /Tm="57.1"
                     /Self_Dimer_Tm="None"
                     /%GC="30.8"
                     /created_by="primer3"
                     /Extension="ATGATGGATATCA"
                     /modified_by="Alex"
                     /label="oAMP126 (MG_Rev)"
     ligation        1040..1069
                     /label="Ligation"
     repeat_region   1311..1345
                     /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(1356..1373)
                     /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(1356..1372)
                     /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(1365..1387)
                     /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(1581..1600)
                     /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    1695..1702
                     /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     1702..1724
                     /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    1751..1757
                     /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(1763..1781)
                     /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        1849..1953
                     /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             1954..2814
                     /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(2172..2191)
                     /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    2669..2674
                     /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    2670
                     /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    2673
                     /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      2985..3573
                     /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     3474..3493
                     /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     3727..3744
                     /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    3753
                     /Original_Bases="A"
                     /label="A"
                     /note="Geneious type: Editing History Replacement"
     misc_feature    3765..3853
                     /Original_Bases
                     /label
                     /note="Geneious type: Editing History Insertion"
     protein_bind    3950..3971
                     /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        3986..4016
                     /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    4024..4040
                     /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     4029..4051
                     /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     4048..4064
                     /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     4048..4064
                     /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   4090..4152
                     /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    4328..4409
                     /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(4526..5125)
                     /created_by="wyssuser"
                     /label="Puromycin"
     misc_feature    4574
                     /Original_Bases="T"
                     /label="T"
                     /note="Geneious type: Editing History Replacement"
     misc_feature    5125^5126
                     /Original_Bases="CCAT"
                     /label="CCAT"
                     /note="Geneious type: Editing History Deletion"
     CDS             complement(5126..5182)
                     /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"
     ligation        <5126..5139
                     /label="Ligation"
     CDS             complement(5192..5935)
                     /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"
                     /label="Tet-On(R) 3G"
     misc_feature    5654
                     /Original_Bases="G"
                     /label="G"
                     /note="Geneious type: Editing History Replacement"
     CDS             complement(5936..5959)
                     /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      5959..5968
                     /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(5988..6199)
                     /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"
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 gcctccgcgg tggcggcatc ggtacacaag tttgtacaaa aaagctgaac gagaaacgta
      421 aaatgatata aatatcaata tattaaatta gattttgcat aaaaaacaga ctacataata
      481 ctgtaaaaca caacatatcc agtcactatg gcgacagaag aagtataggg ataacagggt
      541 aattgttgta agcgcgctat gatggaggct atgccactag aatctgcgtt cgctacctta
      601 ggaccgttat agttagaagg aaagctccat catagtgact ggatatgttg tgttttacag
      661 tattatgtag tctgtttttt atgcaaaatc taatttaata tattgatatt tatatcattt
      721 tacgtttctc gttcagcttt cttgtacaaa gtggtatacc catacgatgt tccagattac
      781 gcttagtaat gagaaatact cagaagatgt cactcaccat agagagacca ttaatgggtc
      841 tcctaggcca ctgagcaaga actatgaatg agatctttag acatattgcg gccgctttct
      901 ccgcgatcca gacatgataa gatacattga tgagtttgga caaaccaaaa ctagaatgca
      961 gtgaaaaaaa tgccttattt gtgaaatttg tgatgctatt gccttatttg taaccattat
     1021 aagctgcaat aaacaagtta tctataacaa gaaaatatat atataataag ttatcacgta
     1081 agtagaacat gaaataacaa tataattatc gtatgagtta aatcttaaaa gtcacgtaaa
     1141 agataatcat gcgtcatttt gactcacgcg gtcgttatag ttcaaaatca gtgacactta
     1201 ccgcattgac aagcacgcct cacgggagct ccaagcggcg actgagatgt cctaaatgca
     1261 cagcgacgga ttcgcgctat ttagaaagag agagcaatat ttcaagaatg catgcgtcaa
     1321 ttttacgcag actatctttc tagggttaag aattcactgg ccgtcgtttt acaacgtcgt
     1381 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     1441 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     1501 aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     1561 cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     1621 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     1681 agacaagctg tgaccatgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     1741 cgaaacacgc gagttcgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     1801 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     1861 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     1921 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     1981 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     2041 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     2101 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     2161 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     2221 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     2281 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     2341 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     2401 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     2461 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     2521 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     2581 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     2641 gctgataaat ctggagccgg tgagcgtggt tcacgcggta tcattgcagc actggggcca
     2701 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     2761 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     2821 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     2881 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     2941 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3001 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     3061 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     3121 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     3181 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     3241 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3301 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3361 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     3421 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     3481 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     3541 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     3601 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     3661 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     3721 gagcgcagcg agtcagtgag cgaggaagcg gatgagcgcc caatacgcaa accgcctctc
     3781 accgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     3841 ggcagtgagc gcaacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     3901 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     3961 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     4021 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagg
     4081 tcgacttaac cctagaaaga taatcatatt gtgacgtacg ttaaagataa tcatgcgtaa
     4141 aattgacgca tgtgttttat cggtctgtat atcgaggttt atttattaat ttgaatagat
     4201 attaagtttt attatattta cacttacata ctaataataa attcaacaaa caatttattt
     4261 atgtttattt atttattaaa aaaaaacaaa aactcaaaat ttcttctata aagtaacaaa
     4321 acttttagca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     4381 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc
     4441 aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta
     4501 tggctgatta tgatcctctg gagattcatg cgccaggttt tctggtcatg caccacgttc
     4561 gtgggccctc aggaacctcc acgtcagcgg taactgtgaa gcccagtcgc tcgtaaaaag
     4621 gaagattccg gggagcactg gtttcgagga acgccggcac gccagcccgt tccgcggcct
     4681 caacaccagg aagaacgaca gcgcttccca aacctttacc ttggtgatct gggctgacac
     4741 cgacagtggc aagaaaccac gcaggctcct ttggtctatg aggagccagc aagccctcca
     4801 tttgctgttg tgccgccaac ctactccctg acagctcggc catccgcgga cctatttccg
     4861 cgaacactgc gccggcttcg acgctttccg gagtagtcca gaccgcaact gcggcaccat
     4921 cgtccgcaac ccaaacttta ccaatgtcca gacccacgcg cgtaagaaac agctcttgca
     4981 actcagttac cctctctatg tgtctgtccg gatctacggt atggcgggtc gctggataat
     5041 ctgcaaatgc tgcggcaagt gttcggacag cccgtggtac atcgtcacgg gtagcgagcc
     5101 taacagtggg cttatattcc gtcatggggc cggggttctc ctccacgtcg ccggcctgct
     5161 tcagcaggct gaagttggtg gcgccgctgc ccccggggag catgtcaagg tcaaaatcgt
     5221 caagagcgtc agcaggcagc atatcaaggt caaagtcgtc aagggcatcg gctgggagca
     5281 tgtctaagtc aaaatcgtca agggcgtcgg tcggcccgcc gctttcgcac tttagctgtt
     5341 tctccaggcc acatatgatt agttccaggc cgaaaaggaa ggcaggttcg gctccctgcc
     5401 ggtcgaacag ctcaattgct tgtttcagaa gtgggggcat agaatcggtg gtaggtgtct
     5461 ctctttcctc ttttgctact tgatgctcct gttcctccaa tacgcagccc agtgtaaagt
     5521 ggcccacggc ggacagagcg tacagtgcgt tctccaggga gaagccttgc tgacacagga
     5581 acgcgagctg attttccagg gtttcgtact gtttctctgt tgggcgggtg ccgagatgca
     5641 ctttagcccc gtcacgatgt gagaggagag cacagcggta tgacttggcg ttgttccgca
     5701 gaaagtcttg ccatgactcg ccttccaggg ggcaggagtg ggtatgatgc ctgtccagca
     5761 tctcgattgg cagggcatcg agcagggccc gcttgttctt cacgtgccag tacagggtag
     5821 gctgctcaac tcccagcttt tgagcgagtt tccttgtcgt caggccttcg ataccgactc
     5881 cattgagtaa ttccagagca gagtttatga ctttgctctt gtccagtcta gacatcttat
     5941 cgtcatcgtc tttgtaatcc atggtggcgg atcccgcgtc acgacacctg tgttctggcg
     6001 gcaaacccgt tgcgaaaaag aacgttcacg gcgactactg cacttatata cggttctccc
     6061 ccaccctcgg gaaaaaggcg gagccagtac acgacatcac tttcccagtt taccccgcgc
     6121 caccttctct aggcaccggt tcaattgccg acccctcccc ccaacttctc ggggactgtg
     6181 ggcgatgtgc gctctgccca ctgacgggca ccggagccac tcgagtggaa tt
//
