LOCUS       PD0055_pIVTRup(original)-MS2-p65-HSF1        5630 bp    DNA     circular UNA 30-JAN-2020
DEFINITION  Concatenation of 2 sequences.
ACCESSION   urn.local...6n-bsz22pg
VERSION     urn.local...6n-bsz22pg
KEYWORDS    .
SOURCE      
  ORGANISM  
            .
FEATURES             Location/Qualifiers
     primer_bind     2..21
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="1388907162197397c5eed9-74a0-47ed-abca-f
                     7e6075ac522"
                     /%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    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="a2b49e31-e190-4bfd-b251-2f761b1f712a"
                     /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..1659
                     /Source="referenceToDocument: urn:local:.:11m-bsz22tq"
                     /label="PCR_MS2 (concatenated sequence 2)"
                     /note="Geneious type: Concatenated sequence"
     CDS             232..234
                     /codon_start=1
                     /product="start codon"
                     /note="ATG"
                     /translation="M"
                     /label="start codon CDS"
     misc_feature    232..255
                     /Sequence="ATGGCTTCAAACTTTACTCAGTTC"
                     /Hairpin_Tm="35.9"
                     /Tm="57.7"
                     /Self_Dimer_Tm="None"
                     /%GC="37.5"
                     /created_by="primer3"
                     /label="mRNA_FWD"
                     /note="Geneious type: primer_bind_reverse"
     primer_bind     232..621
                     /Sequence="ATGGCTTCAAACTTTACTCAGTTCGTGCTCGTGGACAATGGTGGGAC
                     AGGGGATGTGACAGTGGCTCCTTCTAATTTCGCTAATGGGGTGGCAGAGTGGATCAGC
                     TCCAACTCACGGAGCCAGGCCTACAAGGTGACATGCAGCGTCAGGCAGTCTAGTGCCC
                     AGAAGAGAAAGTATACCATCAAGGTGGAGGTCCCCAAAGTGGCTACCCAGACAGTGGG
                     CGGAGTCGAACTGCCTGTCGCCGCTTGGAGGTCCTACCTGAACATGGAGCTCACTATC
                     CCAATTTTCGCTACCAATTCTGACTGTGAACTCATCGTGAAGGCAATGCAGGGGCTCC
                     TCAAAGACGGTAATCCTATCCCTTCCGCCATCGCCGCTAACTCAGGTATCTAC"
                     /Color="#3489bd"
                     /created_by="User"
                     /label="MS2 coat protein"
     primer_bind     232..255
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="735481717479a502da05-133a-4ad9-b568-dd1
                     6f03c6288"
                     /%GC="37.5"
                     /Tm="57.7"
                     /Hairpin_Tm="35.9"
                     /Self_Dimer_Tm="None"
                     /Sequence="ATGGCTTCAAACTTTACTCAGTTC"
                     /label="MS2_FWD"
     CDS             235..621
                     /codon_start=1
                     /product="bacteriophage MS2 coat protein"
                     /note="MS2-N55K"
                     /note="binds with high affinity to a specific stem-loop 
                     structure in the viral RNA (Lim et al., 1994)"
                     /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKV
                     TCSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCE
                     LIVKAMQGLLKDGNPIPSAIAANSGIY"
                     /label="bacteriophage MS2 coat protein CDS"
     primer_bind     563..584
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="138892329399284f32c2a9-cc22-4335-9858-e
                     4636cd227f2"
                     /%GC="54.5"
                     /Tm="60.7"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="GGCTCCTCAAAGACGGTAATCC"
                     /label="PCR_0277"
     primer_bind     622..711
                     /Sequence="AGCGCTGGAGGAGGTGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGG
                     TAGCGGACCTAAGAAAAAGAGGAAGGTGGCGGCCGCTGGATCC"
                     /Color="#c3d9ff"
                     /created_by="User"
                     /label="Linker-NLS"
     modified_base   628..672
                     /created_by="User"
                     /Color="#cdeb8b"
                     /modified_by="User"
                     /label="(GGGGS)3"
     CDS             676..696
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
                     /Color="#6bba70"
                     /modified_by="User"
                     /label="NLS"
     CDS             712..1254
                     /codon_start=1
                     /product="C-terminal portion of the p65 subunit of mouse 
                     NF-kappa-B"
                     /note="p65"
                     /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLT
                     PGPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDN
                     SEFQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSG
                     DEDFSSIADMDFSALLSQISS"
                     /label="C-terminal portion of the p65 subunit of mouse 
                     NF-kappa-B CDS"
     primer_bind     712..1263
                     /Sequence="CCTTCAGGGCAGATCAGCAACCAGGCCCTGGCTCTGGCCCCTAGCTC
                     CGCTCCAGTGCTGGCCCAGACTATGGTGCCCTCTAGTGCTATGGTGCCTCTGGCCCAG
                     CCACCTGCTCCAGCCCCTGTGCTGACCCCAGGACCACCCCAGTCACTGAGCGCTCCAG
                     TGCCCAAGTCTACACAGGCCGGCGAGGGGACTCTGAGTGAAGCTCTGCTGCACCTGCA
                     GTTCGACGCTGATGAGGACCTGGGAGCTCTGCTGGGGAACAGCACCGATCCCGGAGTG
                     TTCACAGATCTGGCCTCCGTGGACAACTCTGAGTTTCAGCAGCTGCTGAATCAGGGCG
                     TGTCCATGTCTCATAGTACAGCCGAACCAATGCTGATGGAGTACCCCGAAGCCATTAC
                     CCGGCTGGTGACCGGCAGCCAGCGGCCCCCCGACCCCGCTCCAACTCCCCTGGGAACC
                     AGCGGCCTGCCTAATGGGCTGTCCGGAGATGAAGACTTCTCAAGCATCGCTGATATGG
                     ACTTTAGTGCCCTGCTGTCACAGATTTCCTCTAGTGGGCAG"
                     /Color="#bb4381"
                     /created_by="User"
                     /label="p65"
     primer_bind     712..736
                     /Mismatches=0
                     /Extension="TTCATCCCGTGAATTC"
                     /created_by="primer3"
                     /annotation_group="194974223378044b22fa92-d085-4e09-9d20-e
                     69f1cff55df"
                     /%GC="60.0"
                     /Tm="67.3"
                     /Hairpin_Tm="35.8"
                     /Self_Dimer_Tm="12.5"
                     /Pair_Dimer_Tm="None"
                     /Sequence="CCTTCAGGGCAGATCAGCAACCAGG"
                     /Product_Size=939
                     /label="PCR_0070"
     primer_bind     712..736
                     /Mismatches=0
                     /Extension="TTCATCCCGTGAATTC"
                     /created_by="primer3"
                     /annotation_group="1388848056919056705d73-c24e-4a90-ad90-7
                     8be168ce695"
                     /%GC="60.0"
                     /Tm="67.3"
                     /Hairpin_Tm="35.8"
                     /Self_Dimer_Tm="12.5"
                     /Sequence="CCTTCAGGGCAGATCAGCAACCAGG"
                     /label="PCR_0070"
     primer_bind     1051..1072
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="13889277136000094c21da-5bf4-4e87-9969-4
                     6d9f4922411"
                     /%GC="50.0"
                     /Tm="59.4"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="TCCATGTCTCATAGTACAGCCG"
                     /label="PCR_0278"
     primer_bind     1054..1076
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="1388885613257258dfd835-55d2-4614-9d08-3
                     ad8c8bc5afe"
                     /%GC="47.8"
                     /Tm="59.9"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="ATGTCTCATAGTACAGCCGAACC"
                     /label="PCR_0117"
     primer_bind     1192..1214
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="13888947337933ea2f501d-35ba-46a8-864b-d
                     6b800b99243"
                     /%GC="47.8"
                     /Tm="60.0"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="16.0"
                     /Sequence="GATGAAGACTTCTCAAGCATCGC"
                     /label="PCR_0121"
     primer_bind     1194..1214
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="13889030772767eabbd88d-f8c2-4ccd-b135-4
                     b42a5731a44"
                     /%GC="47.6"
                     /Tm="58.7"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="16.0"
                     /Sequence="TGAAGACTTCTCAAGCATCGC"
                     /label="PCR_0125"
     primer_bind     1264..1278
                     /Sequence="GGAGGAGGTGGAAGC"
                     /Hairpin_Tm="None"
                     /Tm="51.9"
                     /Self_Dimer_Tm="None"
                     /%GC="66.7"
                     /created_by="primer3"
                     /Color="#cdeb8b"
                     /label="GGGGS"
     CDS             1279..1650
                     /codon_start=1
                     /product="C-terminal activation domain from the human heat
                      shock transcription factor HSF1"
                     /note="HSF1"
                     /translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPP
                     RPPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFA
                     EDPTISLLTGSEPPKAKDPTVS"
                     /label="C-terminal activation domain from the human heat 
                     shock transcription factor HSF1 CDS"
     primer_bind     1279..1650
                     /Sequence="GGCTTCAGCGTGGACACCAGTGCCCTGCTGGACCTGTTCAGCCCCTC
                     GGTGACCGTGCCCGACATGAGCCTGCCTGACCTTGACAGCAGCCTGGCCAGTATCCAA
                     GAGCTCCTGTCTCCCCAGGAGCCCCCCAGGCCTCCCGAGGCAGAGAACAGCAGCCCGG
                     ATTCAGGGAAGCAGCTGGTGCACTACACAGCGCAGCCGCTGTTCCTGCTGGACCCCGG
                     CTCCGTGGACACCGGGAGCAACGACCTGCCGGTGCTGTTTGAGCTGGGAGAGGGCTCC
                     TACTTCTCCGAAGGGGACGGCTTCGCCGAGGACCCCACCATCTCCCTGCTGACAGGCT
                     CGGAGCCTCCCAAAGCCAAGGACCCCACTGTCTCC"
                     /Color="#9999ff"
                     /created_by="User"
                     /label="HSF1"
     primer_bind     1376..1397
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="1388890210473377e8187e-b60e-4d2d-8c97-0
                     2a31edaf5ec"
                     /%GC="54.5"
                     /Tm="59.6"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="10.9"
                     /Sequence="GTATCCAAGAGCTCCTGTCTCC"
                     /label="PCR_0118"
     primer_bind     1376..1397
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="13889316144624584eade1-55e2-4ec6-8c77-7
                     afc753b0ba2"
                     /%GC="54.5"
                     /Tm="59.6"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="10.9"
                     /Sequence="GTATCCAAGAGCTCCTGTCTCC"
                     /label="PCR_0279"
     misc_feature    complement(1626..1650)
                     /Mismatches=0
                     /Extension="GGGACATCAAGGATCC"
                     /created_by="primer3"
                     /annotation_group="194974223378044b22fa92-d085-4e09-9d20-e
                     69f1cff55df"
                     /%GC="60.0"
                     /Tm="67.2"
                     /Hairpin_Tm="38.6"
                     /Self_Dimer_Tm="None"
                     /Pair_Dimer_Tm="None"
                     /Sequence="GGAGACAGTGGGGTCCTTGGCTTTG"
                     /Product_Size=939
                     /label="PCR_0071"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(1626..1650)
                     /Mismatches=0
                     /Extension="GGGACATCAAGGATCC"
                     /created_by="primer3"
                     /annotation_group="13888519109082fa301bca-3ebd-4e97-a5b3-6
                     597ef43e326"
                     /%GC="60.0"
                     /Tm="67.2"
                     /Hairpin_Tm="38.6"
                     /Self_Dimer_Tm="None"
                     /Sequence="GGAGACAGTGGGGTCCTTGGCTTTG"
                     /label="PCR_0071"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(1636..1656)
                     /Sequence="TGTACAGGAGACAGTGGGGTC"
                     /Hairpin_Tm="38.1"
                     /Tm="60.8"
                     /Self_Dimer_Tm="None"
                     /%GC="57.1"
                     /created_by="primer3"
                     /Extension="TCA"
                     /label="mRNA_REV"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(1636..1656)
                     /Mismatches=0
                     /Extension="TCA"
                     /created_by="primer3"
                     /annotation_group="73552897966048df0702-391b-4c38-a58f-7ba
                     8a13ce340"
                     /%GC="57.1"
                     /Tm="60.8"
                     /Hairpin_Tm="38.1"
                     /Self_Dimer_Tm="None"
                     /Sequence="TGTACAGGAGACAGTGGGGTC"
                     /label="MS2_REV"
                     /note="Geneious type: primer_bind_reverse"
     intron          complement(1651..1656)
                     /created_by="User"
                     /label="still_include"
     misc_feature    complement(1657..1659)
                     /label="stop"
     misc_feature    1660..231
                     /Source="referenceToDocument: urn:local:.:11l-bsz22tq"
                     /label="101362 - pIVTRup - EcoRV (concatenated sequence
                     1)"
                     /note="Geneious type: Concatenated sequence"
     misc_feature    1660..1766
                     /Sequence="ATCGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTC
                     TCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGTGAGGGTCT
                     AG"
                     /created_by="User"
                     /label="5'UTR"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    complement(1751..>1766)
                     /Sequence="TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
                     TTTTTTTTCTAGACCCTCACTTCC"
                     /Mismatches=0
                     /annotation_group="b048659d-342d-44f2-b88d-592441d0bd17"
                     /label="pIVTRup-BsmBI-REV"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    <1767..>1821
                     /Source="referenceToDocument: urn:local:.:11l-bsz22tq"
                     /label="101362 - pIVTRup - EcoRV (concatenated sequence
                     1)"
                     /note="Geneious type: Concatenated sequence"
     3'UTR           1767..1821
                     /created_by="User"
                     /label="polyA(55)"
     misc_feature    <1767..>1821
                     /Source="referenceToDocument: urn:local:.:11n-bsz22tq"
                     /label="pIVTRup-linearized (concatenated sequence 1)"
                     /note="Geneious type: Concatenated sequence"
     misc_feature    1821^1822
                     /Original_Bases="AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAAAAAAAAAAA"
                     /label="AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
                     AAAAAAAAAAAAAAAAA"
                     /note="Geneious type: Editing History Deletion"
     primer_bind     complement(1936..1952)
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /label="M13 rev"
     primer_bind     complement(1936..1952)
                     /note="In lacZ gene. Also called M13-rev"
                     /label="M13 Reverse"
     primer_bind     complement(1949..1971)
                     /note="In lacZ gene"
                     /label="M13/pUC Reverse"
     protein_bind    1960..1976
                     /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     1978..1998
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="138891949814519501bd2c-f444-4766-93a8-1
                     ee7e4296579"
                     /%GC="57.1"
                     /Tm="62.7"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="TCCACACAACATACGAGCCGG"
                     /label="PCR_0227"
     primer_bind     1979..1996
                     /Mismatches=0
                     /Extension="ACTGGCGAACTACTTACTCTAGCTTCCCGG"
                     /created_by="primer3"
                     /annotation_group="138886452349260d7395ae-2c34-4910-9d8d-6
                     d4aceabfc19"
                     /%GC="55.6"
                     /Tm="55.8"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="CCACACAACATACGAGCC"
                     /label="PCR_0096"
     primer_bind     1979..1996
                     /Mismatches=0
                     /Extension="TGTGGTGATGTTGAAGAAAACCCTGGTCCA"
                     /created_by="primer3"
                     /annotation_group="13888733600537690c854d-8f6f-438c-8ea7-2
                     8cb24fb2dc5"
                     /%GC="55.6"
                     /Tm="55.8"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="CCACACAACATACGAGCC"
                     /label="PCR_0099"
     promoter        complement(1984..2014)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     misc_feature    complement(2001..2021)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="1388898945428748f45171-3bbd-4c59-8d7e-e
                     5628655ce6b"
                     /%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    2029..2050
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                      of cAMP."
                     /label="CAP binding site"
     misc_feature    complement(2163..2182)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="1388855927833845ced462-3621-403b-9be9-5
                     7f98c631415"
                     /%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(2163..2182)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="13889152113284b515464b-378c-4c02-98a6-c
                     017a6007f59"
                     /%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(2167..2184)
                     /note="L4440 vector, forward primer"
                     /label="L4440"
     misc_feature    complement(2266..2286)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="13888775191020c6f29d41-5238-4085-adc7-c
                     7ac3e8cd758"
                     /%GC="52.4"
                     /Tm="60.1"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="GCTCACATGTTCTTTCCTGCG"
                     /label="PCR_0101"
                     /note="Geneious type: primer_bind_reverse"
     rep_origin      complement(2338..2926)
                     /direction="LEFT"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /label="ori"
     misc_feature    complement(2378..2397)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="13889112656740371bd3d2-f26f-4698-9582-7
                     8856b55e4d8"
                     /%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(2418..2437)
                     /note="pBR322 origin, forward primer"
                     /label="pBR322ori-F"
     CDS             complement(3097..3957)
                     /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"
     primer_bind     3250..3270
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="13889397432220ca68d801-3869-496b-ba2b-1
                     27125406473"
                     /%GC="52.4"
                     /Tm="60.2"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="ACCGGCTCCAGATTTATCAGC"
                     /label="qPCR_002"
     misc_feature    complement(3324..3351)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="138883515032229b3c39ed-f2b0-43d3-90f2-c
                     2cfafff8680"
                     /%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(3324..3351)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="13888437995184a175c0a4-7404-4cbb-9f6c-b
                     24048edaa2e"
                     /%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(3325..3351)
                     /Mismatches=0
                     /Extension="GGCGAAAGGGGGATGTGCTGCAAGGCGATT"
                     /created_by="primer3"
                     /annotation_group="13888691048325decdbd71-35c0-4ae8-8957-1
                     908a07ee3b5"
                     /%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     3352..3380
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="13888307150495226fb20a-9c81-4919-9713-0
                     610db78e5ca"
                     /%GC="55.2"
                     /Tm="67.7"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="CCGGGAAGCTAGAGTAAGTAGTTCGCCAG"
                     /label="PCR_0015"
     primer_bind     3352..3380
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="138883956258888518f54a-4ab5-4165-924e-5
                     be3882fa690"
                     /%GC="55.2"
                     /Tm="67.7"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="CCGGGAAGCTAGAGTAAGTAGTTCGCCAG"
                     /label="PCR_0067"
     misc_feature    complement(3433..3453)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="1388935599654238205ff9-4525-459c-a54f-0
                     39940886941"
                     /%GC="52.4"
                     /Tm="59.3"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Sequence="GAAGCCATACCAAACGACGAG"
                     /label="qPCR_001"
                     /note="Geneious type: primer_bind_reverse"
     primer_bind     3720..3739
                     /note="Ampicillin resistance gene, reverse primer"
                     /label="Amp-R"
     CDS             complement(3975..4769)
                     /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(4086..4105)
                     /note="Neomycin resistance gene, forward primer"
                     /label="Neo-F"
     primer_bind     4696..4715
                     /note="Neomycin resistance gene, reverse primer"
                     /label="Neo-R"
     rep_origin      complement(5113..5541)
                     /direction="LEFT"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     primer_bind     complement(5223..5244)
                     /note="F1 origin, forward primer"
                     /label="F1ori-F"
     primer_bind     5435..5454
                     /note="F1 origin, reverse primer"
                     /label="F1ori-R"
     primer_bind     5470..5488
                     /modified_by="User"
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="138888166128098cceab60-43cf-42bd-9e13-2
                     71daaf94c8a"
                     /%GC="57.9"
                     /Tm="60.3"
                     /Hairpin_Tm="36.2"
                     /Self_Dimer_Tm="None"
                     /Sequence="TGGCAAGTGTAGCGGTCAC"
                     /label="PCR_0114"
     misc_feature    complement(5623..13)
                     /Mismatches=0
                     /created_by="primer3"
                     /annotation_group="13888602177981e11b309c-d9ea-4884-8d3b-0
                     de3baec3a2d"
                     /%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 tatggcttca
      241 aactttactc agttcgtgct cgtggacaat ggtgggacag gggatgtgac agtggctcct
      301 tctaatttcg ctaatggggt ggcagagtgg atcagctcca actcacggag ccaggcctac
      361 aaggtgacat gcagcgtcag gcagtctagt gcccagaaga gaaagtatac catcaaggtg
      421 gaggtcccca aagtggctac ccagacagtg ggcggagtcg aactgcctgt cgccgcttgg
      481 aggtcctacc tgaacatgga gctcactatc ccaattttcg ctaccaattc tgactgtgaa
      541 ctcatcgtga aggcaatgca ggggctcctc aaagacggta atcctatccc ttccgccatc
      601 gccgctaact caggtatcta cagcgctgga ggaggtggaa gcggaggagg aggaagcgga
      661 ggaggaggta gcggacctaa gaaaaagagg aaggtggcgg ccgctggatc cccttcaggg
      721 cagatcagca accaggccct ggctctggcc cctagctccg ctccagtgct ggcccagact
      781 atggtgccct ctagtgctat ggtgcctctg gcccagccac ctgctccagc ccctgtgctg
      841 accccaggac caccccagtc actgagcgct ccagtgccca agtctacaca ggccggcgag
      901 gggactctga gtgaagctct gctgcacctg cagttcgacg ctgatgagga cctgggagct
      961 ctgctgggga acagcaccga tcccggagtg ttcacagatc tggcctccgt ggacaactct
     1021 gagtttcagc agctgctgaa tcagggcgtg tccatgtctc atagtacagc cgaaccaatg
     1081 ctgatggagt accccgaagc cattacccgg ctggtgaccg gcagccagcg gccccccgac
     1141 cccgctccaa ctcccctggg aaccagcggc ctgcctaatg ggctgtccgg agatgaagac
     1201 ttctcaagca tcgctgatat ggactttagt gccctgctgt cacagatttc ctctagtggg
     1261 cagggaggag gtggaagcgg cttcagcgtg gacaccagtg ccctgctgga cctgttcagc
     1321 ccctcggtga ccgtgcccga catgagcctg cctgaccttg acagcagcct ggccagtatc
     1381 caagagctcc tgtctcccca ggagcccccc aggcctcccg aggcagagaa cagcagcccg
     1441 gattcaggga agcagctggt gcactacaca gcgcagccgc tgttcctgct ggaccccggc
     1501 tccgtggaca ccgggagcaa cgacctgccg gtgctgtttg agctgggaga gggctcctac
     1561 ttctccgaag gggacggctt cgccgaggac cccaccatct ccctgctgac aggctcggag
     1621 cctcccaaag ccaaggaccc cactgtctcc tgtacatgaa tcgctgcctt ctgcggggct
     1681 tgccttctgg ccatgccctt cttctctccc ttgcacctgt acctcttggt ctttgaataa
     1741 agcctgagta ggaagtgagg gtctagaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     1801 aaaaaaaaaa aaaaaaaaaa acgatcaata accgacactc ctgcgtcgcc tacgcgctac
     1861 gatctgcaga attccagcac actggcggcc gttactagtg gatccgagct cggtaccaag
     1921 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     1981 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     2041 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     2101 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     2161 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     2221 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     2281 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     2341 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     2401 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     2461 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     2521 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     2581 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     2641 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     2701 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     2761 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     2821 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     2881 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     2941 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     3001 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     3061 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     3121 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     3181 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     3241 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     3301 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     3361 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     3421 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     3481 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     3541 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     3601 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     3661 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     3721 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     3781 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     3841 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     3901 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     3961 cttccttttt caattcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa
     4021 tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct
     4081 tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg
     4141 ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca
     4201 tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag cctggcgaac
     4261 agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg
     4321 gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag
     4381 gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg
     4441 gcaggagcaa ggtgggatga caggagatcc tgccccggca cttcgcccaa tagcagccag
     4501 tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc
     4561 agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga caggtcggtc
     4621 ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag
     4681 ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa
     4741 cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca
     4801 gatcttgatc ccctgcgcca tcagatcctt ggcggcaaga aagccatcca gtttactttg
     4861 cagggcttcc caaccttacc agagggcgcc ccagctggca attccggttc gcttgctgtc
     4921 cataaaaccg cccagtctag ctatcgccat gtaagcccac tgcaagctac ctgctttctc
     4981 tttgcgcttg cgttttccct tgtccagata gcccagtagc tgacattcat ccggggtcag
     5041 caccgtttct gcggactggc tttctacgtg ttccgcttcc tttagcagcc cttgcgccct
     5101 gaattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     5161 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     5221 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     5281 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     5341 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     5401 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     5461 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     5521 atgcgccgct acagggcgcg tccattcgcc attcaggctg cgcaactgtt gggaagggcg
     5581 atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
//
