LOCUS       PD0053_pIVTRup(original)-EBFP2        4922 bp    DNA     circular UNA 30-JAN-2020
DEFINITION  Concatenation of 2 sequences.
ACCESSION   urn.local...6l-bsz22pf
VERSION     urn.local...6l-bsz22pf
KEYWORDS    .
SOURCE      
  ORGANISM  
            .
FEATURES             Location/Qualifiers
     primer_bind     2..21
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="13450965307250a842458e-ca79-4d40-a446-d
                     c61e781f2cf"
                     /%GC="50.0"
                     /Tm="58.8"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="TGCAAGGCGATTAAGTTGGG"
                     /label="PCR_0128"
     primer_bind     37..59
                     /note="In lacZ gene"
                     /label="M13/pUC Forward"
     primer_bind     51..68
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
                     /label="M13 Forward"
     primer_bind     52..68
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /label="M13 fwd"
     promoter        75..93
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /label="T7 promoter"
     misc_feature    101..106
                     /Recognition_pattern="GGGCC^C"
                     /annotation_group="GGGCCC"
                     /label="ApaI"
                     /note="Geneious type: restriction site"
     misc_feature    101..106
                     /Recognition_pattern="G^GGCCC"
                     /annotation_group="GGGCCC"
                     /label="Bsp120I, PspOMI"
                     /note="Geneious type: restriction site"
     misc_feature    112..117
                     /Recognition_pattern="ATGCA^T"
                     /annotation_group="ATGCAT"
                     /label="EcoT22I, Mph1103I, NsiI, Zsp2I"
                     /note="Geneious type: restriction site"
     misc_feature    118..125
                     /Recognition_pattern="VC^TCGAGB"
                     /annotation_group="VCTCGAGB"
                     /label="PspXI"
                     /note="Geneious type: restriction site"
     misc_feature    119..124
                     /Recognition_pattern="C^TCGAG"
                     /annotation_group="CTCGAG"
                     /label="XhoI, PaeR7I, Sfr274I, SlaI"
                     /note="Geneious type: restriction site"
     misc_feature    124..131
                     /Recognition_pattern="GC^GGCCGC"
                     /annotation_group="GCGGCCGC"
                     /label="NotI, CciNI"
                     /note="Geneious type: restriction site"
     misc_feature    145..205
                     /Sequence="TTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAG
                     AGAGAAAAGAAGAG"
                     /created_by="User"
                     /label="pIVTRup_FWD"
                     /note="Geneious type: primer_bind_reverse"
     primer_bind     145..205
                     /Sequence="TTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAG
                     AGAGAAAAGAAGAG"
                     /Mismatches=0
                     /annotation_group="6ebb3ecc-8b7b-43bb-ae38-626da2f8bb04"
                     /label="pIVTRup-BsmBI-FWD"
     promoter        165..183
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /label="T7 promoter"
     misc_feature    182..231
                     /Sequence="GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC
                     GAT"
                     /created_by="User"
                     /label="3'UTR"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    223..228
                     /Sequence="GCCACC"
                     /Hairpin_Tm="None"
                     /Tm="2.1"
                     /Self_Dimer_Tm="None"
                     /%GC="83.3"
                     /created_by="primer3"
                     /Color="#c3d9ff"
                     /modified_by="User"
                     /label="Kozak"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    232..951
                     /Source="referenceToDocument: urn:local:.:11c-bsz22tp"
                     /label="PCR_EBFP2 (concatenated sequence 2)"
                     /note="Geneious type: Concatenated sequence"
     misc_feature    232..252
                     /Sequence="ATGGTGAGCAAGGGCGAGGAG"
                     /Hairpin_Tm="47.5"
                     /Tm="64.5"
                     /Self_Dimer_Tm="None"
                     /%GC="61.9"
                     /created_by="primer3"
                     /label="mRNA_FWD"
                     /note="Geneious type: primer_bind_reverse"
     primer_bind     232..252
                     /Mismatches=0
                     /Extension="GGATCCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGCGACG
                     TGGAAGAAAACCCCGGTCCT"
                     /%GC="61.9"
                     /Hairpin_Tm="47.5"
                     /Self_Dimer_Tm="None"
                     /Tm="64.5"
                     /annotation_group="2511354876301481ab0afd-0885-4427-9980-7
                     55d21ed03cd"
                     /Sequence="ATGGTGAGCAAGGGCGAGGAG"
                     /created_by="primer3"
                     /label="PCR_0062"
     primer_bind     232..252
                     /Sequence="ATGGTGAGCAAGGGCGAGGAG"
                     /Hairpin_Tm="47.5"
                     /Tm="64.5"
                     /Self_Dimer_Tm="None"
                     /%GC="61.9"
                     /modified_by="ptraxler"
                     /label="PCR_0058"
     primer_bind     232..252
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="536023438904ae7fe8da-096e-47ba-b8f5-a5a
                     0ba0c02b4"
                     /%GC="61.9"
                     /Tm="64.5"
                     /Hairpin_Tm="47.5"
                     /Self_Dimer_Tm="None"
                     /Sequence="ATGGTGAGCAAGGGCGAGGAG"
                     /label="mCherry/EBFP2_FWD"
     primer_bind     232..252
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="134504643954699a86417c-05b7-42fe-9acc-4
                     d126fe2cf20"
                     /%GC="61.9"
                     /Tm="64.5"
                     /Hairpin_Tm="47.5"
                     /Self_Dimer_Tm="None"
                     /Sequence="ATGGTGAGCAAGGGCGAGGAG"
                     /label="PCR_0058"
     primer_bind     232..252
                     /Mismatches=0
                     /Extension="GGATCCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGCGACG
                     TGGAAGAAAACCCCGGTCCT"
                     /created_by="primer3"
                     /annotation_group="13450503629842e7cc9652-56fb-48d0-abcb-4
                     ba8142cc1c7"
                     /%GC="61.9"
                     /Tm="64.5"
                     /Hairpin_Tm="47.5"
                     /Self_Dimer_Tm="None"
                     /Sequence="ATGGTGAGCAAGGGCGAGGAG"
                     /label="PCR_0062"
     misc_feature    247..252
                     /Recognition_pattern="GAGGAG(10/8)"
                     /annotation_group="GAGGAG"
                     /label="BseRI"
                     /note="Geneious type: restriction site"
     misc_feature    258..265
                     /Recognition_pattern="CR^CCGGYG"
                     /annotation_group="CRCCGGYG"
                     /label="SgrAI"
                     /note="Geneious type: restriction site"
     misc_feature    complement(295..300)
                     /Recognition_pattern="CACGTC(-3/-3)"
                     /annotation_group="CACGTC"
                     /label="BtrI, AjiI, BmgBI"
                     /note="Geneious type: restriction site"
     misc_feature    414..420
                     /Recognition_pattern="G^GTNACC"
                     /annotation_group="GGTNACC"
                     /label="BstEII, BstPI, Eco91I, EcoO65I, PspEI"
                     /note="Geneious type: restriction site"
     misc_feature    648..658
                     /Recognition_pattern="CCANNNN^NTGG"
                     /annotation_group="CCANNNNNTGG"
                     /label="PflMI, AccB7I, Van91I"
                     /note="Geneious type: restriction site"
     misc_feature    complement(682..693)
                     /gene="DNTT"
                     /gene_synonym="TDT"
                     /experiment="experimental evidence, no additional details
                     recorded"
                     /note="Region: Deoxynucleoside triphosphate binding.
                     {ECO:0000250|UniProtKB:P09838}; propagated from
                     UniProtKB/Swiss-Prot (P04053.3)"
                     /X-Transferred_From="NM_001017520.1_Iso2_shorter;
                     NM_004088.3_TdT_SV40NLS_2xTY1tag;
                     NM_004088.3_TdT_SV40NLS_GGS;
                     NM_004088.3_TdT_SV40NLS_GGS_2xTY1tag;
                     NM_004088.3_TdT_SV40NLS_GGS_HAtag;
                     NM_004088.3_iso1_long_hTdTS (short isoform of
                     hTdT)_acc.to.Troshchynsky2015; NM_004088.3_iso1_long_hTdTS
                     (short isoform of hTdT)_acc.to.Troshchynsky2015 - DNTT
                     CDS"
                     /X-Transferred_Similarity="100.00%"
                     /Transferred_From="gBlock_TdT PCR Product"
                     /Transferred_Similarity="100.00%"
                     /label="DNTT misc"
     misc_feature    852..858
                     /Recognition_pattern="GC^TNAGC"
                     /annotation_group="GCTNAGC"
                     /label="BlpI, Bpu1102I, Bsp1720I"
                     /note="Geneious type: restriction site"
     misc_feature    860..866
                     /Recognition_pattern="RG^GWCCY"
                     /annotation_group="RGGWCCY"
                     /label="PpuMI, Psp5II, PspPPI"
                     /note="Geneious type: restriction site"
     misc_feature    923..929
                     /Recognition_pattern="CC^CWGGG"
                     /annotation_group="CCCWGGG"
                     /label="PasI"
                     /note="Geneious type: restriction site"
     misc_feature    complement(928..951)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="134504247487959a2db513-e61c-49d8-a540-b
                     6bcb4f0b690"
                     /%GC="54.2"
                     /Tm="64.9"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="TCACTTGTACAGCTCGTCCATGCC"
                     /label="PCR_0054"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(928..948)
                     /Sequence="CTTGTACAGCTCGTCCATGCC"
                     /Hairpin_Tm="None"
                     /Tm="61.3"
                     /Self_Dimer_Tm="None"
                     /%GC="57.1"
                     /created_by="primer3"
                     /Extension="TCA"
                     /label="mRNA_REV"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(928..948)
                     /Mismatches=0
                     /Extension="TCA"
                     /created_by="primer3"
                     /annotation_group="5359732364000f6a1aa7-c40a-4972-a9a2-c00
                     4809c521d"
                     /%GC="57.1"
                     /Tm="61.3"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="CTTGTACAGCTCGTCCATGCC"
                     /label="EBFP2_REV"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    941..946
                     /Recognition_pattern="T^GTACA"
                     /annotation_group="TGTACA"
                     /label="Bsp1407I, BsrGI, BstAUI"
                     /note="Geneious type: restriction site"
     misc_feature    952..231
                     /Source="referenceToDocument: urn:local:.:11b-bsz22tp"
                     /label="101362 - pIVTRup - EcoRV (concatenated sequence
                     1)"
                     /note="Geneious type: Concatenated sequence"
     misc_feature    952..1058
                     /Sequence="ATCGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTC
                     TCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGTGAGGGTCT
                     AG"
                     /created_by="User"
                     /label="5'UTR"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(1043..>1058)
                     /Sequence="TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
                     TTTTTTTTCTAGACCCTCACTTCC"
                     /Mismatches=0
                     /annotation_group="736b6cd2-f05c-4d0d-b9d7-c2d89f686941"
                     /label="pIVTRup-BsmBI-REV"
                     /note="Geneious type: primer_bind_reverse"
     3'UTR           1059..1113
                     /created_by="User"
                     /label="polyA(55)"
     misc_feature    <1059..>1113
                     /Source="referenceToDocument: urn:local:.:11d-bsz22tp"
                     /label="pIVTRup-linearized (concatenated sequence 1)"
                     /note="Geneious type: Concatenated sequence"
     misc_feature    1113^1114
                     /Original_Bases="AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAAAAAAAAAAA"
                     /label="AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAA"
                     /note="Geneious type: Editing History Deletion"
     misc_feature    1161..1166
                     /Recognition_pattern="G^AATTC"
                     /annotation_group="GAATTC"
                     /label="EcoRI"
                     /note="Geneious type: restriction site"
     misc_feature    1186..1191
                     /Recognition_pattern="A^CTAGT"
                     /annotation_group="ACTAGT"
                     /label="SpeI, AhlI, BcuI"
                     /note="Geneious type: restriction site"
     misc_feature    1192..1197
                     /Recognition_pattern="G^GATCC"
                     /annotation_group="GGATCC"
                     /label="BamHI"
                     /note="Geneious type: restriction site"
     misc_feature    1198..1203
                     /Recognition_pattern="GAG^CTC"
                     /annotation_group="GAGCTC"
                     /label="Ecl136II, Eco53kI, EcoICRI"
                     /note="Geneious type: restriction site"
     misc_feature    1198..1203
                     /Recognition_pattern="GAGCT^C"
                     /annotation_group="GAGCTC"
                     /label="SacI, Psp124BI, SstI"
                     /note="Geneious type: restriction site"
     misc_feature    1210..1215
                     /Recognition_pattern="A^AGCTT"
                     /annotation_group="AAGCTT"
                     /label="HindIII"
                     /note="Geneious type: restriction site"
     primer_bind     complement(1228..1244)
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /label="M13 rev"
     primer_bind     complement(1228..1244)
                     /note="In lacZ gene. Also called M13-rev"
                     /label="M13 Reverse"
     primer_bind     complement(1241..1263)
                     /note="In lacZ gene"
                     /label="M13/pUC Reverse"
     protein_bind    1252..1268
                     /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)."
                     /label="lac operator"
     primer_bind     1270..1290
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="1345109657223546b5763a-d254-42b2-8b6f-d
                     5b6a7aabc12"
                     /%GC="57.1"
                     /Tm="62.7"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="TCCACACAACATACGAGCCGG"
                     /label="PCR_0227"
     primer_bind     1271..1288
                     /Mismatches=0
                     /Extension="ACTGGCGAACTACTTACTCTAGCTTCCCGG"
                     /created_by="primer3"
                     /annotation_group="134507178355075cd2907a-ada1-4569-b58c-1
                     e60b0a5e441"
                     /%GC="55.6"
                     /Tm="55.8"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="CCACACAACATACGAGCC"
                     /label="PCR_0096"
     primer_bind     1271..1288
                     /Mismatches=0
                     /Extension="TGTGGTGATGTTGAAGAAAACCCTGGTCCA"
                     /created_by="primer3"
                     /annotation_group="13450800497735635741c5-ab0e-4fe2-82ca-0
                     a4e9057a955"
                     /%GC="55.6"
                     /Tm="55.8"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="CCACACAACATACGAGCC"
                     /label="PCR_0099"
     promoter        complement(1276..1306)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     misc_feature    complement(1293..1313)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="1345092537895887410b4a-3e45-4ae9-976f-d
                     aeff4c35062"
                     /%GC="47.6"
                     /Tm="57.2"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="CCCAGGCTTTACACTTTATGC"
                     /label="PCR_0122"
                     /note="Geneious type: primer_bind_reverse"
     protein_bind    1321..1342
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                      of cAMP."
                     /label="CAP binding site"
     misc_feature    complement(1455..1474)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="134506269287280b223c08-3c18-48dc-bcd0-9
                     78914c88648"
                     /%GC="60.0"
                     /Tm="60.2"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="9.1"
                     /Sequence="CGAGTCAGTGAGCGAGGAAG"
                     /label="PCR_0083"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(1455..1474)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="1345105511080867edb325-8db6-4524-9dd6-1
                     2aecc610c44"
                     /%GC="60.0"
                     /Tm="60.2"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="9.1"
                     /Sequence="CGAGTCAGTGAGCGAGGAAG"
                     /label="PCR_0179"
                     /note="Geneious type: primer_bind_reverse"
     primer_bind     complement(1459..1476)
                     /note="L4440 vector, forward primer"
                     /label="L4440"
     misc_feature    complement(1558..1578)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="134508432658671ed9e7bf-8e10-4e33-ba83-f
                     05b486fd91f"
                     /%GC="52.4"
                     /Tm="60.1"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="GCTCACATGTTCTTTCCTGCG"
                     /label="PCR_0101"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    1569..1574
                     /Recognition_pattern="A^CATGT"
                     /annotation_group="ACATGT"
                     /label="PciI, PscI"
                     /note="Geneious type: restriction site"
     rep_origin      complement(1630..2218)
                     /direction="LEFT"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /label="ori"
     misc_feature    complement(1670..1689)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="13451011758403e116ecd6-e97b-4356-b444-0
                     964779ac7b4"
                     /%GC="60.0"
                     /Tm="60.7"
                     /Hairpin_Tm="36.7"
                     /Self_Dimer_Tm="None"
                     /Sequence="CACCTCTGACTTGAGCGTCG"
                     /label="PCR_0143"
                     /note="Geneious type: primer_bind_reverse"
     primer_bind     complement(1710..1729)
                     /note="pBR322 origin, forward primer"
                     /label="pBR322ori-F"
     misc_feature    2289..2294
                     /Recognition_pattern="T^CATGA"
                     /annotation_group="TCATGA"
                     /label="BspHI, CciI, PagI"
                     /note="Geneious type: restriction site"
     CDS             complement(2389..3249)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="confers resistance to ampicillin, carbenicillin,
                     and related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW"
                     /label="AmpR"
     misc_feature    2457..2467
                     /Recognition_pattern="GACNNN^NNGTC"
                     /annotation_group="GACNNNNNGTC"
                     /label="Eam1105I, AhdI, BmeRI, DriI"
                     /note="Geneious type: restriction site"
     misc_feature    complement(2529..2534)
                     /Recognition_pattern="GGTCTC(1/5)"
                     /annotation_group="GGTCTC"
                     /label="Eco31I, BsaI, Bso31I, BspTNI"
                     /note="Geneious type: restriction site"
     primer_bind     2542..2562
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="13451175900975726a7a34-cfb9-4cbc-989b-d
                     a3eb6a9d28d"
                     /%GC="52.4"
                     /Tm="60.2"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="ACCGGCTCCAGATTTATCAGC"
                     /label="qPCR_002"
     misc_feature    complement(2616..2643)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="13450382412615710f3435-1087-45fd-b1b0-d
                     de5cc56753e"
                     /%GC="46.4"
                     /Tm="63.8"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="CAACAATTAATAGACTGGATGGAGGCGG"
                     /label="PCR_0020"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(2616..2643)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="13450585449252e34d0270-fa56-4ab9-a07f-2
                     bace671e50a"
                     /%GC="46.4"
                     /Tm="63.8"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="CAACAATTAATAGACTGGATGGAGGCGG"
                     /label="PCR_0068"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(2617..2643)
                     /Mismatches=0
                     /Extension="GGCGAAAGGGGGATGTGCTGCAAGGCGATT"
                     /created_by="primer3"
                     /annotation_group="134507580806175c5fe8ca-d86b-4ed8-bfbf-4
                     22c161623c2"
                     /%GC="44.4"
                     /Tm="62.1"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="CAACAATTAATAGACTGGATGGAGGCG"
                     /label="PCR_0097"
                     /note="Geneious type: primer_bind_reverse"
     primer_bind     2644..2672
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="13450341925248ffac9647-445a-4cba-ac29-1
                     02299d63d7c"
                     /%GC="55.2"
                     /Tm="67.7"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="CCGGGAAGCTAGAGTAAGTAGTTCGCCAG"
                     /label="PCR_0015"
     primer_bind     2644..2672
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="13450545123267042adf1f-d319-484e-9410-2
                     613228c4659"
                     /%GC="55.2"
                     /Tm="67.7"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="CCGGGAAGCTAGAGTAAGTAGTTCGCCAG"
                     /label="PCR_0067"
     misc_feature    complement(2725..2745)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="1345113656276347bcebc9-4106-49a9-abde-5
                     9f34f775734"
                     /%GC="52.4"
                     /Tm="59.3"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="GAAGCCATACCAAACGACGAG"
                     /label="qPCR_001"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    2940..2945
                     /Recognition_pattern="AGT^ACT"
                     /annotation_group="AGTACT"
                     /label="ScaI, BmcAI, ZrmI"
                     /note="Geneious type: restriction site"
     primer_bind     3012..3031
                     /note="Ampicillin resistance gene, reverse primer"
                     /label="Amp-R"
     misc_feature    3057..3066
                     /Recognition_pattern="GAANN^NNTTC"
                     /annotation_group="GAANNNNTTC"
                     /label="XmnI, Asp700I, MroXI, PdmI"
                     /note="Geneious type: restriction site"
     CDS             complement(3267..4061)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /note="confers resistance to neomycin, kanamycin, and G418
                      (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
                     PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
                     LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY
                     QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
                     /label="NeoR/KanR"
     primer_bind     complement(3378..3397)
                     /note="Neomycin resistance gene, forward primer"
                     /label="Neo-F"
     misc_feature    3412..3418
                     /Recognition_pattern="CG^GWCCG"
                     /annotation_group="CGGWCCG"
                     /label="RsrII, CpoI, CspI, Rsr2I"
                     /note="Geneious type: restriction site"
     misc_feature    3496..3501
                     /Recognition_pattern="C^CATGG"
                     /annotation_group="CCATGG"
                     /label="NcoI, Bsp19I"
                     /note="Geneious type: restriction site"
     misc_feature    3531..3536
                     /Recognition_pattern="G^CGCGC"
                     /annotation_group="GCGCGC"
                     /label="BsePI, BssHII, PauI, PteI"
                     /note="Geneious type: restriction site"
     misc_feature    3810..3818
                     /Recognition_pattern="GACN^NNGTC"
                     /annotation_group="GACNNNGTC"
                     /label="Tth111I, PflFI, PsyI"
                     /note="Geneious type: restriction site"
     primer_bind     3988..4007
                     /note="Neomycin resistance gene, reverse primer"
                     /label="Neo-R"
     misc_feature    4087..4092
                     /Recognition_pattern="T^GATCA"
                     /annotation_group="TGATCA"
                     /label="BclI, FbaI, Ksp22I"
                     /note="Geneious type: restriction site"
     misc_feature    4092..4097
                     /Recognition_pattern="A^GATCT"
                     /annotation_group="AGATCT"
                     /label="BglII"
                     /note="Geneious type: restriction site"
     misc_feature    4110..4124
                     /Recognition_pattern="CCANNNNN^NNNNTGG"
                     /annotation_group="CCANNNNNNNNNTGG"
                     /label="XcmI"
                     /note="Geneious type: restriction site"
     misc_feature    4377..4387
                     /Recognition_pattern="GCANNNN^NTGC"
                     /annotation_group="GCANNNNNTGC"
                     /label="BstAPI"
                     /note="Geneious type: restriction site"
     rep_origin      complement(4405..4833)
                     /direction="LEFT"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     misc_feature    4472..4477
                     /Recognition_pattern="TTA^TAA"
                     /annotation_group="TTATAA"
                     /label="PsiI, AanI"
                     /note="Geneious type: restriction site"
     primer_bind     complement(4515..4536)
                     /note="F1 origin, forward primer"
                     /label="F1ori-F"
     misc_feature    4544..4556
                     /Recognition_pattern="(7/12)GAACNNNNNNTCC(12/7)"
                     /annotation_group="GAACNNNNNNTCC"
                     /label="AloI"
                     /note="Geneious type: restriction site"
     primer_bind     4727..4746
                     /note="F1 origin, reverse primer"
                     /label="F1ori-R"
     primer_bind     4762..4780
                     /modified_by="User"
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="13450887284350999d9d63-c7f3-4a73-8751-3
                     d5fede970f2"
                     /%GC="57.9"
                     /Tm="60.3"
                     /Hairpin_Tm="36.2"
                     /Self_Dimer_Tm="None"
                     /Sequence="TGGCAAGTGTAGCGGTCAC"
                     /label="PCR_0114"
     misc_feature    complement(4915..13)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="1345066903856529718a39-6c25-432f-b5d3-e
                     00e8db08262"
                     /%GC="57.1"
                     /Tm="63.9"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="AATCGCCTTGCAGCACATCCC"
                     /label="PCR_0093"
                     /note="Geneious type: primer_bind_reverse"
ORIGIN      
        1 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
       61 cggccagtga attgtaatac gactcactat agggcgaatt gggccctcta gatgcatgct
      121 cgagcggccg ccagtgtgat ggatttggac cctcgtacag aagctaatac gactcactat
      181 agggaaataa gagagaaaag aagagtaaga agaaatataa gagccaccga tatggtgagc
      241 aagggcgagg agctgttcac cggcgtggtg cccatcctgg tggagctgga cggcgacgtg
      301 aacggccaca agttcagcgt gaggggcgag ggcgagggcg acgccaccaa cggcaagctg
      361 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctggtgacc
      421 accctgagcc acggcgtgca gtgcttcgcc aggtaccccg accacatgaa gcagcacgac
      481 ttcttcaaga gcgccatgcc cgagggctac gtgcaggaga ggaccatctt cttcaaggac
      541 gacggcacct acaagaccag ggccgaggtg aagttcgagg gcgacaccct ggtgaacagg
      601 atcgagctga agggcgtgga cttcaaggag gacggcaaca tcctgggcca caagctggag
      661 tacaacttca acagccacaa catctacatc atggccgtga agcagaagaa cggcatcaag
      721 gtgaacttca agatcaggca caacgtggag gacggcagcg tgcagctggc cgaccactac
      781 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacagcca ctacctgagc
      841 acccagagcg tgctgagcaa ggaccccaac gagaagaggg accacatggt gctgctggag
      901 ttcaggaccg ccgccggcat caccctgggc atggacgagc tgtacaagtg aatcgctgcc
      961 ttctgcgggg cttgccttct ggccatgccc ttcttctctc ccttgcacct gtacctcttg
     1021 gtctttgaat aaagcctgag taggaagtga gggtctagaa aaaaaaaaaa aaaaaaaaaa
     1081 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaacgatcaa taaccgacac tcctgcgtcg
     1141 cctacgcgct acgatctgca gaattccagc acactggcgg ccgttactag tggatccgag
     1201 ctcggtacca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     1261 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     1321 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     1381 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     1441 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     1501 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     1561 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     1621 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     1681 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     1741 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     1801 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     1861 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     1921 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     1981 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     2041 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     2101 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     2161 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     2221 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     2281 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     2341 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     2401 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     2461 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     2521 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     2581 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     2641 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     2701 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     2761 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     2821 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     2881 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     2941 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     3001 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     3061 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     3121 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     3181 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     3241 aatactcata ctcttccttt ttcaattcag aagaactcgt caagaaggcg atagaaggcg
     3301 atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg
     3361 ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc
     3421 acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc
     3481 ggcaagcagg catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg
     3541 agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga
     3601 tcgacaagac cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg
     3661 tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg
     3721 gatactttct cggcaggagc aaggtgggat gacaggagat cctgccccgg cacttcgccc
     3781 aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg
     3841 cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg
     3901 gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg
     3961 gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa
     4021 gcggccggag aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct
     4081 gtctcttgat cagatcttga tcccctgcgc catcagatcc ttggcggcaa gaaagccatc
     4141 cagtttactt tgcagggctt cccaacctta ccagagggcg ccccagctgg caattccggt
     4201 tcgcttgctg tccataaaac cgcccagtct agctatcgcc atgtaagccc actgcaagct
     4261 acctgctttc tctttgcgct tgcgttttcc cttgtccaga tagcccagta gctgacattc
     4321 atccggggtc agcaccgttt ctgcggactg gctttctacg tgttccgctt cctttagcag
     4381 cccttgcgcc ctgaattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     4441 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     4501 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     4561 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     4621 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     4681 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     4741 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     4801 ccgccgcgct taatgcgccg ctacagggcg cgtccattcg ccattcaggc tgcgcaactg
     4861 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
     4921 tg
//
