LOCUS       pAMP536_(PB-CT3G-ERP2-M 6656 bp ds-DNA     circular     10-JUN-2024
DEFINITION  an expression clone created from pAMP449 (PB-CT3G-ERP2-MG-BC) and
            pAMP425 (pDONR™221 - UBB).
ACCESSION   urn.local...1c3-d3zz2il
VERSION     urn.local...1c3-d3zz2il
COMMENT     Imported using the Genbank importer. File name: pAMP536
            (PB-CT3G-ERP2-MG-BC-UBB).gb
FEATURES             Location/Qualifiers
     promoter        9..262
                     /label="TRE3GS promoter"
                     /ApEinfo_revcolor="#346ee0"
                     /ApEinfo_fwdcolor="#346ee0"
                     /note="3rd-generation Tet-responsive promoter that can beactivated by binding of Tet-On(R) 3G, modified toeliminate binding sites for endogenous mammaliantranscription factors"
                     /locus_tag="TRE3GS promoter"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="TRE3GS promoter"
     protein_bind    17..35
                     /label="tet operator"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /gene="tetO"
                     /bound_moiety="tetracycline repressor TetR"
                     /locus_tag="tet operator"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="tet operator"
     protein_bind    53..71
                     /label="tet operator"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /gene="tetO"
                     /bound_moiety="tetracycline repressor TetR"
                     /locus_tag="tet operator(1)"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="tet operator(1)"
     protein_bind    89..107
                     /label="tet operator"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /gene="tetO"
                     /bound_moiety="tetracycline repressor TetR"
                     /locus_tag="tet operator(2)"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="tet operator(2)"
     protein_bind    125..143
                     /label="tet operator"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /gene="tetO"
                     /bound_moiety="tetracycline repressor TetR"
                     /locus_tag="tet operator(3)"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="tet operator(3)"
     protein_bind    161..179
                     /label="tet operator"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /gene="tetO"
                     /bound_moiety="tetracycline repressor TetR"
                     /locus_tag="tet operator(4)"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="tet operator(4)"
     protein_bind    197..215
                     /label="tet operator"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /gene="tetO"
                     /bound_moiety="tetracycline repressor TetR"
                     /locus_tag="tet operator(5)"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="tet operator(5)"
     protein_bind    233..251
                     /label="tet operator"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /gene="tetO"
                     /bound_moiety="tetracycline repressor TetR"
                     /locus_tag="tet operator(6)"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="tet operator(6)"
     source          263..264
                     /label="source:synthetic DNA construct"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
                     /locus_tag="source:synthetic DNA construct(2)"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="source:synthetic DNA construct(2)"
     promoter        265..364
                     /label="mCMV Promoter"
                     /ApEinfo_revcolor="#b1ff67"
                     /ApEinfo_fwdcolor="#b1ff67"
                     /created_by="wyssuser"
     misc_feature    265..364
                     /label="mCMV promoter"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /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%"
     misc            368..385
                     /label="Spacer"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
                     /Color=-1
     source          381..385
                     /label="source:synthetic DNA construct"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
                     /locus_tag="source:synthetic DNA construct(2)"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="source:synthetic DNA construct(2)"
     attB1           387..407
                     /label="attB1"
                     /ApEinfo_revcolor="#faac61"
                     /ApEinfo_fwdcolor="#faac61"
                     /annotation_group="attB"
     misc            413..418
                     /label="Kozak"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
                     /Color=-1
     primer_bind     419..440
                     /label="oASL331 (UBB_Fwd)"
                     /ApEinfo_revcolor="#9eafd2"
                     /ApEinfo_fwdcolor="#9eafd2"
                     /Sequence="ATGCAGATCTTCGTGAAAACCC"
                     /Hairpin_Tm="None"
                     /Tm="59.3"
                     /Self_Dimer_Tm="None"
                     /GC="45.5"
                     /Extension="GGGGACAAGTTTGTACAAAAAAGCAGGCTTAGCCACC"
                     /modified_by="Alex"
     CDS             419..1108
                     /label="UBB"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /created_by="Alex"
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGC*"
     primer_bind     complement(1090..1108)
                     /label="oASL332 (UBB_Rev)"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
                     /Sequence="TTAACAGCCACCCCTCAGG"
                     /Hairpin_Tm="43.8"
                     /Tm="58.9"
                     /Self_Dimer_Tm="None"
                     /GC="57.9"
                     /created_by="primer3"
                     /Extension="GGGGACCACTTTGTACAAGAAAGCTGGGTA"
     attB2           complement(1113..1133)
                     /label="attB2"
                     /ApEinfo_revcolor="#f8d3a9"
                     /ApEinfo_fwdcolor="#f8d3a9"
                     /annotation_group="attB"
     misc_feature    1135..1135
                     /ApEinfo_revcolor="#ffef86"
                     /ApEinfo_fwdcolor="#ffef86"
                     /note="Geneious type: Editing History Insertion"
                     /Original_Bases=""
     CDS             1136..1162
                     /label="HA Tag"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /created_by="wyssuser"
                     /translation="YPYDVPDYA"
     terminator      1163..1171
                     /label="source:synthetic DNA construct"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
                     /locus_tag="source:synthetic DNA construct(4)"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /modified_by="wyssuser"
                     /label="STOPS"
     primer_bind     1175..1197
                     /label="BC_Fwd"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /Sequence="ATACTCAGAAGATGTCACTCACC"
                     /Hairpin_Tm="None"
                     /Tm="57.4"
                     /Self_Dimer_Tm="None"
                     /GC="43.5"
                     /created_by="primer3"
                     /Extension="ACACTCTTTCCCTACACGACGCTCTTCCGATCTNN"
                     /modified_by="Alex"
     misc_difference 1202..1224
                     /label="Barcode linker 5'"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /created_by="wyssuser"
     misc_feature    1202..1267
                     /label="ACACCGGGTCTTCGAGAAGACCTGTT"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
                     /note="Geneious type: Editing History Replacement"
                     /Original_Bases="ACACCGGGTCTTCGAGAAGACCTGTT"
     misc_feature    1225..1244
                     /label="20mer Barcode"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
     misc_difference complement(1245..1267)
                     /label="Barcode linker 3'"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /created_by="wyssuser"
                     /modified_by="wyssuser"
     primer_bind     complement(1272..1294)
                     /label="BC_Rev"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /Sequence="CATTCATAGTTCTTGCTCAGTGG"
                     /Hairpin_Tm="None"
                     /Tm="57.5"
                     /Self_Dimer_Tm="None"
                     /GC="43.5"
                     /created_by="primer3"
                     /Extension="GAGTTCAGACGTGTGCTCTTCCGATCTNNN"
                     /modified_by="Alex"
     misc_feature    1295..1300
                     /label="GCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCC"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
                     /note="Geneious type: Editing History Replacement"
                     /Original_Bases="GCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCC"
     misc_feature    1303..1309
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
                     /note="Geneious type: Editing History Insertion"
                     /Original_Bases=""
     misc_feature    1312..1319
                     /ApEinfo_revcolor="#9eafd2"
                     /ApEinfo_fwdcolor="#9eafd2"
                     /note="Geneious type: Editing History Insertion"
                     /Original_Bases=""
     primer_bind     complement(1313..1328)
                     /label="ATTR2-Right-FP modular (274 R) - PolyA"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
                     /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-f1ef6b1429e2"
                     /Sequence="GCGGAGAAAGCGGCCG"
                     /created_by="primer3"
     primer_bind     complement(1325..1346)
                     /label="oAMP271 (BC_Rev)"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /Sequence="TCTTATCATGTCTGGATCGCGG"
                     /Hairpin_Tm="None"
                     /Tm="60.0"
                     /Self_Dimer_Tm="6.1"
                     /GC="50.0"
                     /modified_by="Alex"
     primer_bind     1329..1354
                     /label="PolyA (1 F) - ATTR2-Right-FP modular"
                     /ApEinfo_revcolor="#d59687"
                     /ApEinfo_fwdcolor="#d59687"
                     /_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-b167-7808cb75bb1f"
                     /Sequence="GATCCAGACATGATAAGATACATTGA"
                     /created_by="primer3"
     polyA_signal    1329..1463
                     /label="SV40 poly(A) signal"
                     /ApEinfo_revcolor="#ff3eee"
                     /ApEinfo_fwdcolor="#ff3eee"
                     /note="SV40 polyadenylation signal"
                     /locus_tag="SV40 poly(A) signal"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(1437..1463)
                     /label="PolyA (135 R) - CK461-PB-cT3G-cERP2-Puro BaseVector #4-CMV_Enhancer - mCMV-Tet-Promoter (digested byEcoRV, BbsI) Fragment 4"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /_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-e154a3a53574"
                     /Sequence="AACTTGTTTATTGCAGCTTATAATGGT"
                     /created_by="primer3"
     primer_bind     complement(1438..1463)
                     /label="oAMP126 (MG_Rev)"
                     /ApEinfo_revcolor="#b1ff67"
                     /ApEinfo_fwdcolor="#b1ff67"
                     /Sequence="AACTTGTTTATTGCAGCTTATAATGG"
                     /Hairpin_Tm="None"
                     /Tm="57.1"
                     /Self_Dimer_Tm="None"
                     /GC="30.8"
                     /created_by="primer3"
                     /Extension="ATGATGGATATCA"
                     /modified_by="Alex"
     repeat_region   1735..1769
                     /label="piggyBac left (5') inverted repeat"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
                     /note="piggyBac transposon-specific inverted terminalrepeat sequence (ITR)"
                     /locus_tag="piggyBac left (5') inverted repeat"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="piggyBac left (5') inverted repeat"
     primer_bind     complement(1780..1796)
                     /label="M13 fwd"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
                     /note="common sequencing primer, one of multiple similarvariants"
                     /locus_tag="M13 fwd"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="M13 fwd"
     primer_bind     complement(1780..1797)
                     /label="M13 Forward"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21)Forward"
                     /locus_tag="M13 Forward"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="M13 Forward"
     primer_bind     complement(1789..1811)
                     /label="M13/pUC Forward"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
                     /note="In lacZ gene"
                     /locus_tag="M13/pUC Forward"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="M13/pUC Forward"
     primer_bind     complement(2005..2024)
                     /label="pRS-marker"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
                     /note="pRS vectors, use to sequence yeast selectablemarker"
                     /locus_tag="pRS-marker"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="pRS-marker"
     misc_feature    2119..2126
                     /label="bsmbi"
                     /ApEinfo_revcolor="#fb0106"
                     /ApEinfo_fwdcolor="#fb0106"
                     /locus_tag="bsmbi"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="bsmbi"
     primer_bind     2126..2148
                     /label="pGEX 3'"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
                     /note="pGEX vectors, reverse primer"
                     /locus_tag="pGEX 3'"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="pGEX 3'"
     misc_feature    2175..2181
                     /label="bsmbi"
                     /ApEinfo_revcolor="#fb0106"
                     /ApEinfo_fwdcolor="#fb0106"
                     /locus_tag="bsmbi(1)"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="bsmbi(1)"
     primer_bind     complement(2187..2205)
                     /label="pBRforEco"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
                     /note="pBR322 vectors, upsteam of EcoRI site, forwardprimer"
                     /locus_tag="pBRforEco"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="pBRforEco"
     promoter        2273..2377
                     /label="AmpR promoter"
                     /ApEinfo_revcolor="#346ee0"
                     /ApEinfo_fwdcolor="#346ee0"
                     /gene="bla"
                     /locus_tag="AmpR promoter"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="AmpR promoter"
     CDS             2378..3238
                     /label="AmpR"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_fwdcolor="#e9d024"
                     /note="confers resistance to ampicillin, carbenicillin,and related antibiotics"
                     /gene="bla"
                     /product="beta-lactamase"
                     /locus_tag="AmpR"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="AmpR"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW*"
     primer_bind     complement(2596..2615)
                     /label="Amp-R"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
                     /note="Ampicillin resistance gene, reverse primer"
                     /locus_tag="Amp-R"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="Amp-R"
     misc_feature    3093..3098
                     /label="bsaI"
                     /ApEinfo_revcolor="#fb0106"
                     /ApEinfo_fwdcolor="#fb0106"
                     /locus_tag="bsaI"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="bsaI"
     misc_feature    3094..3094
                     /label="recoded"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
                     /locus_tag="recoded"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="recoded"
     misc_feature    3097..3097
                     /label="recoded"
                     /ApEinfo_revcolor="#6666fe"
                     /ApEinfo_fwdcolor="#6666fe"
                     /locus_tag="recoded(1)"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="recoded(1)"
     rep_origin      3409..3997
                     /label="ori"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin ofreplication"
                     /direction="RIGHT"
                     /locus_tag="ori"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="ori"
     primer_bind     3898..3917
                     /label="pBR322ori-F"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
                     /note="pBR322 origin, forward primer"
                     /locus_tag="pBR322ori-F"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="pBR322ori-F"
     primer_bind     4151..4168
                     /label="L4440"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
                     /note="L4440 vector, forward primer"
                     /locus_tag="L4440"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="L4440"
     misc_feature    4177..4177
                     /label="A"
                     /ApEinfo_revcolor="#b1ff67"
                     /ApEinfo_fwdcolor="#b1ff67"
                     /note="Geneious type: Editing History Replacement"
                     /Original_Bases="A"
     misc_feature    4189..4277
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
                     /note="Geneious type: Editing History Insertion"
                     /Original_Bases=""
     protein_bind    4374..4395
                     /label="CAP binding site"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /note="CAP binding activates transcription in thepresenceof cAMP."
                     /bound_moiety="E. coli catabolite activator protein"
                     /locus_tag="CAP binding site"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="CAP binding site"
     promoter        4410..4440
                     /label="lac promoter"
                     /ApEinfo_revcolor="#346ee0"
                     /ApEinfo_fwdcolor="#346ee0"
                     /note="promoter for the E. coli lac operon"
                     /locus_tag="lac promoter"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="lac promoter"
     protein_bind    4448..4464
                     /label="lac operator"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /note="The lac repressor binds to the lac operator toinhibit transcription in E. coli. This inhibition can berelieved by adding lactose orisopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /bound_moiety="lac repressor encoded by lacI"
                     /locus_tag="lac operator"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="lac operator"
     primer_bind     4453..4475
                     /label="M13/pUC Reverse"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
                     /note="In lacZ gene"
                     /locus_tag="M13/pUC Reverse"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="M13/pUC Reverse"
     primer_bind     4472..4488
                     /label="M13 rev"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
                     /note="common sequencing primer, one of multiple similarvariants"
                     /locus_tag="M13 rev"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="M13 rev"
     primer_bind     4472..4488
                     /label="M13 Reverse"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
                     /note="In lacZ gene. Also called M13-rev"
                     /locus_tag="M13 Reverse"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="M13 Reverse"
     repeat_region   4514..4576
                     /label="piggyBac right (3') inverted repeat"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
                     /note="piggyBac transposon-specific inverted terminalrepeat sequence (ITR)"
                     /locus_tag="piggyBac right (3') inverted repeat"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="piggyBac right (3') inverted repeat"
     polyA_signal    4752..4833
                     /label="SV40 poly(A) signal"
                     /ApEinfo_revcolor="#ff3eee"
                     /ApEinfo_fwdcolor="#ff3eee"
                     /note="SV40 polyadenylation signal"
                     /locus_tag="SV40 poly(A) signal(1)"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="SV40 poly(A) signal(1)"
     CDS             complement(4950..5549)
                     /label="Puromycin"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /created_by="wyssuser"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA*"
     misc_feature    4998..4998
                     /label="T"
                     /ApEinfo_revcolor="#faac61"
                     /ApEinfo_fwdcolor="#faac61"
                     /note="Geneious type: Editing History Replacement"
                     /Original_Bases="T"
     misc_feature    5549..5550
                     /label="CCAT"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /note="Geneious type: Editing History Deletion"
                     /Original_Bases="CCAT"
     CDS             complement(5550..5606)
                     /label="P2A"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_fwdcolor="#e9d024"
                     /note="Eukaryotic ribosomes fail to insert a peptide bondbetween the Gly and Pro residues, yielding separatepolypeptides."
                     /product="2A peptide from porcine teschovirus-1polyprotein"
                     /locus_tag="P2A"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="P2A"
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(5616..6359)
                     /label="Tet-On(R) 3G"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_fwdcolor="#e9d024"
                     /product="modified rtTA protein that binds tightly topromoters containing the tet operator in the presence ofdoxycycline"
                     /locus_tag="Tet-On(R) 3G"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /Original_Translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA DALDDFDLDMLPADALDDFDLDMLPG"
                     /label="Tet-On(R) 3G"
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPADALDDFDLDMLPADALDDFDLDMLPG"
     misc_feature    6078..6078
                     /label="G"
                     /ApEinfo_revcolor="#b1ff67"
                     /ApEinfo_fwdcolor="#b1ff67"
                     /note="Geneious type: Editing History Replacement"
                     /Original_Bases="G"
     CDS             complement(6360..6383)
                     /label="FLAG"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_fwdcolor="#e9d024"
                     /product="FLAG(R) epitope tag, followed by anenterokinasecleavage site"
                     /locus_tag="FLAG"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="FLAG"
                     /translation="DYKDDDDK"
     regulatory      6383..6392
                     /label="vertebrate consensus sequence for stronginitiation of translation (Kozak, 1987)"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /note="vertebrate consensus sequence for stronginitiationof translation (Kozak, 1987)"
                     /regulatory_class="other"
                     /locus_tag="vertebrate consensus sequence for stronginitiation of translation (Kozak, 1987)"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}width 5 offset 0"
                     /label="vertebrate consensus sequence for stronginitiation of translation (Kozak, 1987)"
     promoter        complement(6412..6623)
                     /label="EF-1-alpha core promoter"
                     /ApEinfo_revcolor="#346ee0"
                     /ApEinfo_fwdcolor="#346ee0"
                     /note="core promoter for human elongation factorEF-1-alpha"
                     /locus_tag="EF-1-alpha core promoter"
                     /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 aaagcaggct tagccaccat
      421 gcagatcttc gtgaaaaccc ttaccggcaa gaccatcacc cttgaggtgg agcccagtga
      481 caccatcgaa aatgtgaagg ccaagatcca ggataaggaa ggcattcccc ccgaccagca
      541 gaggctcatc tttgcaggca agcagctgga agatggccgt actctttctg actacaacat
      601 tcagaaggag tcgaccctgc acctggtcct gcgtctgaga ggtggtatgc agatcttcgt
      661 gaagaccctg accggcaaga ccatcaccct ggaagtggag cccagtgaca ccatcgaaaa
      721 tgtgaaggcc aagatccagg ataaagaagg catccctccc gaccagcaga ggctcatctt
      781 tgcaggcaag cagctggaag atggccgcac tctttctgac tacaacatcc agaaggagtc
      841 gaccctgcac ctggtcctgc gtctgagagg tggtatgcag atcttcgtga agaccctgac
      901 cggcaagacc atcactctgg aggtggagcc cagtgacacc atcgaaaatg tgaaggccaa
      961 gatccaagat aaagaaggca tcccccccga ccagcagagg ctcatctttg caggcaagca
     1021 gctggaagat ggccgcactc tttctgacta caacatccag aaagagtcga ccctgcacct
     1081 ggtcctgcgc ctgaggggtg gctgttaata cccagctttc ttgtacaaag tggtataccc
     1141 atacgatgtt ccagattacg cttagtaatg agaaatactc agaagatgtc actcaccata
     1201 gtccgcagtg aaagataaat gatcTGAATC ACAGAAGAAT ACTCgtttta gagctagaaa
     1261 tagcaagtag gccactgagc aagaactatg aatgagatct ttagacatat tgcggccgct
     1321 ttctccgcga tccagacatg ataagataca ttgatgagtt tggacaaacc aaaactagaa
     1381 tgcagtgaaa aaaatgcctt atttgtgaaa tttgtgatgc tattgcctta tttgtaacca
     1441 ttataagctg caataaacaa gttatctata acaagaaaat atatatataa taagttatca
     1501 cgtaagtaga acatgaaata acaatataat tatcgtatga gttaaatctt aaaagtcacg
     1561 taaaagataa tcatgcgtca ttttgactca cgcggtcgtt atagttcaaa atcagtgaca
     1621 cttaccgcat tgacaagcac gcctcacggg agctccaagc ggcgactgag atgtcctaaa
     1681 tgcacagcga cggattcgcg ctatttagaa agagagagca atatttcaag aatgcatgcg
     1741 tcaattttac gcagactatc tttctagggt taagaattca ctggccgtcg ttttacaacg
     1801 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     1861 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     1921 cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     1981 acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     2041 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     2101 ttacagacaa gctgtgacca tgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     2161 tcaccgaaac acgcgagttc gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     2221 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     2281 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     2341 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     2401 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     2461 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     2521 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     2581 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     2641 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     2701 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     2761 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     2821 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     2881 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     2941 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     3001 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     3061 tattgctgat aaatctggag ccggtgagcg tggttcacgc ggtatcattg cagcactggg
     3121 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     3181 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     3241 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     3301 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     3361 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     3421 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     3481 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     3541 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     3601 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     3661 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     3721 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     3781 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     3841 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     3901 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     3961 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4021 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     4081 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     4141 gaccgagcgc agcgagtcag tgagcgagga agcggatgag cgcccaatac gcaaaccgcc
     4201 tctcaccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     4261 agcgggcagt gagcgcaacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     4321 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     4381 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     4441 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
     4501 aaggtcgact taaccctaga aagataatca tattgtgacg tacgttaaag ataatcatgc
     4561 gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag gtttatttat taatttgaat
     4621 agatattaag ttttattata tttacactta catactaata ataaattcaa caaacaattt
     4681 atttatgttt atttatttat taaaaaaaaa caaaaactca aaatttcttc tataaagtaa
     4741 caaaactttt agcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     4801 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     4861 tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt
     4921 ggtatggctg attatgatcc tctggagatt catgcgccag gttttctggt catgcaccac
     4981 gttcgtgggc cctcaggaac ctccacgtca gcggtaactg tgaagcccag tcgctcgtaa
     5041 aaaggaagat tccggggagc actggtttcg aggaacgccg gcacgccagc ccgttccgcg
     5101 gcctcaacac caggaagaac gacagcgctt cccaaacctt taccttggtg atctgggctg
     5161 acaccgacag tggcaagaaa ccacgcaggc tcctttggtc tatgaggagc cagcaagccc
     5221 tccatttgct gttgtgccgc caacctactc cctgacagct cggccatccg cggacctatt
     5281 tccgcgaaca ctgcgccggc ttcgacgctt tccggagtag tccagaccgc aactgcggca
     5341 ccatcgtccg caacccaaac tttaccaatg tccagaccca cgcgcgtaag aaacagctct
     5401 tgcaactcag ttaccctctc tatgtgtctg tccggatcta cggtatggcg ggtcgctgga
     5461 taatctgcaa atgctgcggc aagtgttcgg acagcccgtg gtacatcgtc acgggtagcg
     5521 agcctaacag tgggcttata ttccgtcatg gggccggggt tctcctccac gtcgccggcc
     5581 tgcttcagca ggctgaagtt ggtggcgccg ctgcccccgg ggagcatgtc aaggtcaaaa
     5641 tcgtcaagag cgtcagcagg cagcatatca aggtcaaagt cgtcaagggc atcggctggg
     5701 agcatgtcta agtcaaaatc gtcaagggcg tcggtcggcc cgccgctttc gcactttagc
     5761 tgtttctcca ggccacatat gattagttcc aggccgaaaa ggaaggcagg ttcggctccc
     5821 tgccggtcga acagctcaat tgcttgtttc agaagtgggg gcatagaatc ggtggtaggt
     5881 gtctctcttt cctcttttgc tacttgatgc tcctgttcct ccaatacgca gcccagtgta
     5941 aagtggccca cggcggacag agcgtacagt gcgttctcca gggagaagcc ttgctgacac
     6001 aggaacgcga gctgattttc cagggtttcg tactgtttct ctgttgggcg ggtgccgaga
     6061 tgcactttag ccccgtcacg atgtgagagg agagcacagc ggtatgactt ggcgttgttc
     6121 cgcagaaagt cttgccatga ctcgccttcc agggggcagg agtgggtatg atgcctgtcc
     6181 agcatctcga ttggcagggc atcgagcagg gcccgcttgt tcttcacgtg ccagtacagg
     6241 gtaggctgct caactcccag cttttgagcg agtttccttg tcgtcaggcc ttcgataccg
     6301 actccattga gtaattccag agcagagttt atgactttgc tcttgtccag tctagacatc
     6361 ttatcgtcat cgtctttgta atccatggtg gcggatcccg cgtcacgaca cctgtgttct
     6421 ggcggcaaac ccgttgcgaa aaagaacgtt cacggcgact actgcactta tatacggttc
     6481 tcccccaccc tcgggaaaaa ggcggagcca gtacacgaca tcactttccc agtttacccc
     6541 gcgccacctt ctctaggcac cggttcaatt gccgacccct ccccccaact tctcggggac
     6601 tgtgggcgat gtgcgctctg cccactgacg ggcaccggag ccactcgagt ggaatt
//