LOCUS       Exported                8511 bp ds-DNA     circular SYN 17-AUG-2020
DEFINITION  CMV51p> Halotag7-HVR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8511)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Aug 19, 2020 from SnapGene Viewer 5.0.8
            https://www.snapgene.com
COMMENT     Entry Clone:   C453-E04, 2834 bps DNA circular (attL4, attL5 sites)
            Entry Clone:   C514-E47, 3623 bps DNA circular (attR5, attR1 sites)
            Entry Clone:   R703-E05, 2877 bps DNA circular (attL1, attL2 sites)
            Destination Vector:   pDest303, 8412 bps DNA circular (attR4, attR2 
            sites)
            Recombination using LR clonase to produce Expression Clone
FEATURES             Location/Qualifiers
     source          1..8511
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      204..1993
                     /label=oriP
                     /note="Epstein-Barr virus oriP replication origin (Yates et
                     al., 2000)"
     protein_bind    2199..2219
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     enhancer        2230..2533
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2534..2737
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     protein_bind    complement(2805..2825)
                     /gene="mutant version of attB"
                     /label=attB5
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     CDS             2829..3860
                     /codon_start=1
                     /label=Halotag-Hs.KRAS4bHVR
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISGEPTTEDLYFQSDNAIATTLYKKVGKEKMSKDGKKKKKKSKTKC
                     VIM"
     CDS             2829..3719
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             3732..3752
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     protein_bind    3768..3792
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3792..3860
                     /codon_start=1
                     /label=Hs.KRAS4b-HVR
                     /translation="KEKMSKDGKKKKKKSKTKCVIM"
     protein_bind    complement(3977..4001)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(4063..4081)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     polyA_signal    4107..4331
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4377..4805
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        4819..5149
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5000..5135
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5216..6010
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    6184..6305
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6354..6370)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6378..6394
                     /label=lac operator
                     /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)."
     promoter        complement(6402..6432)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6447..6468
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(6756..7344)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7515..8375)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(8376..8480)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatccgcc tttatgtgta actcttggct gaagctctta caccaatgct
       61 gggggacatg tacctcccag gggcccagga agactacggg aggctacacc aacgtcaatc
      121 agaggggcct gtgtagctac cgataagcgg accctcaaga gggcattagc aatagtgttt
      181 ataaggcccc cttgttaacc ctaaacgggt agcatatgct tcccgggtag tagtatatac
      241 tatccagact aaccctaatt caatagcata tgttacccaa cgggaagcat atgctatcga
      301 attagggtta gtaaaagggt cctaaggaac agcgatatct cccaccccat gagctgtcac
      361 ggttttattt acatggggtc aggattccac gagggtagtg aaccatttta gtcacaaggg
      421 cagtggctga agatcaagga gcgggcagtg aactctcctg aatcttcgcc tgcttcttca
      481 ttctccttcg tttagctaat agaataactg ctgagttgtg aacagtaagg tgtatgtgag
      541 gtgctcgaaa acaaggtttc aggtgacgcc cccagaataa aatttggacg gggggttcag
      601 tggtggcatt gtgctatgac accaatataa ccctcacaaa ccccttgggc aataaatact
      661 agtgtaggaa tgaaacattc tgaatatctt taacaataga aatccatggg gtggggacaa
      721 gccgtaaaga ctggatgtcc atctcacacg aatttatggc tatgggcaac acataatcct
      781 agtgcaatat gatactgggg ttattaagat gtgtcccagg cagggaccaa gacaggtgaa
      841 ccatgttgtt acactctatt tgtaacaagg ggaaagagag tggacgccga cagcagcgga
      901 ctccactggt tgtctctaac acccccgaaa attaaacggg gctccacgcc aatggggccc
      961 ataaacaaag acaagtggcc actctttttt ttgaaattgt ggagtggggg cacgcgtcag
     1021 cccccacacg ccgccctgcg gttttggact gtaaaataag ggtgtaataa cttggctgat
     1081 tgtaaccccg ctaaccactg cggtcaaacc acttgcccac aaaaccacta atggcacccc
     1141 ggggaatacc tgcataagta ggtgggcggg ccaagatagg ggcgcgattg ctgcgatctg
     1201 gaggacaaat tacacacact tgcgcctgag cgccaagcac agggttgttg gtcctcatat
     1261 tcacgaggtc gctgagagca cggtgggcta atgttgccat gggtagcata tactacccaa
     1321 atatctggat agcatatgct atcctaatct atatctgggt agcataggct atcctaatct
     1381 atatctgggt agcatatgct atcctaatct atatctgggt agtatatgct atcctaattt
     1441 atatctgggt agcataggct atcctaatct atatctgggt agcatatgct atcctaatct
     1501 atatctgggt agtatatgct atcctaatct gtatccgggt agcatatgct atcctaatag
     1561 agattagggt agtatatgct atcctaattt atatctgggt agcatatact acccaaatat
     1621 ctggatagca tatgctatcc taatctatat ctgggtagca tatgctatcc taatctatat
     1681 ctgggtagca taggctatcc taatctatat ctgggtagca tatgctatcc taatctatat
     1741 ctgggtagta tatgctatcc taatttatat ctgggtagca taggctatcc taatctatat
     1801 ctgggtagca tatgctatcc taatctatat ctgggtagta tatgctatcc taatctgtat
     1861 ccgggtagca tatgctatcc tcatgcatat acagtcagca tatgataccc agtagtagag
     1921 tgggagtgct atcctttgca tatgccgcca cctcccaagg gggcgtgaat tttcgctgct
     1981 tgtccttttc ctgctgggga tctcccgatc ccctatggtg cactctcagt acaatctgct
     2041 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
     2101 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
     2161 atctgcttag ggttaggcgt tttgcgctgc ttcgatcaca actttgtata gaaaagttgg
     2221 ccctgcaggc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
     2281 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
     2341 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
     2401 tcatatgcca agtccgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
     2461 tgcccagtac atgaccttac gggactttcc tacttggcag tacatctacg tattagtcat
     2521 cgctattacc atggtgatgc ggttttggca gtacaccaat gggcgtggat agcggtttga
     2581 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
     2641 aaatcaacgg gactttccaa aatgtcgtaa taaccccgcc ccgttgacgc aaatgggcgg
     2701 taggcgtgta cggtgggagg tctatataag cagagctcgt ttagtgaacc gtcagatcgc
     2761 ctggagacgc catccacgct gttttgacct ccatagaaga cagccaactt ttgtatacaa
     2821 agtggaccat ggcagaaatc ggtactggct ttccattcga cccccattat gtggaagtcc
     2881 tgggcgagcg catgcactac gtcgatgttg gtccgcgcga tggcacccct gtgctgttcc
     2941 tgcacggtaa cccgacctcc tcctacgtgt ggcgcaacat catcccgcat gttgcaccga
     3001 cccatcgctg cattgctcca gacctgatcg gtatgggcaa atccgacaaa ccagacctgg
     3061 gttatttctt cgacgaccac gtccgcttca tggatgcctt catcgaagcc ctgggtctgg
     3121 aagaggtcgt cctggtcatt cacgactggg gctccgctct gggtttccac tgggccaagc
     3181 gcaatccaga gcgcgtcaaa ggtattgcat ttatggagtt catccgccct atcccgacct
     3241 gggacgaatg gccagaattt gcccgcgaga ccttccaggc cttccgcacc accgacgtcg
     3301 gccgcaagct gatcatcgat cagaacgttt ttatcgaggg tacgctgccg atgggtgtcg
     3361 tccgcccgct gactgaagtc gagatggacc attaccgcga gccgttcctg aatcctgttg
     3421 accgcgagcc actgtggcgc ttcccaaacg agctgccaat cgccggtgag ccagcgaaca
     3481 tcgtcgcgct ggtcgaagaa tacatggact ggctgcacca gtcccctgtc ccgaagctgc
     3541 tgttctgggg caccccaggc gttctgatcc caccggccga agccgctcgc ctggccaaaa
     3601 gcctgcctaa ctgcaaggct gtggacatcg gcccgggtct gaatctgctg caagaagaca
     3661 acccggacct gatcggcagc gagatcgcgc gctggctgtc gacgctcgag atttccggcg
     3721 agccaaccac tgaggatctg tactttcaga gcgataacgc gatcgccaca actttgtaca
     3781 aaaaagttgg caaagaaaag atgagcaaag atggtaaaaa gaagaaaaag aagtcaaaga
     3841 caaagtgtgt aattatgtaa gctaactgaa tagagaaaaa aatcactgga tataccaccg
     3901 ttgatatatc ccaatggcat cgtaaagaac attttgaggc atttcagtca gttgctcaat
     3961 gtacctataa ccagaccgtc aactttcttg tacaaagttg tgaaattctg cagatatcca
     4021 tcacactggc ggccgctcga gcatgcatct agagggccct attctatagt gtcacctaaa
     4081 tgctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     4141 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     4201 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     4261 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     4321 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     4381 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     4441 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     4501 cgccggcttt ccccgtcaag ctctaaatcg gggcatccct ttagggttcc gatttagtgc
     4561 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     4621 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     4681 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     4741 gattttgggg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     4801 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccaggc
     4861 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     4921 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     4981 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     5041 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct
     5101 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg
     5161 agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat
     5221 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     5281 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     5341 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga
     5401 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     5461 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     5521 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     5581 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     5641 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     5701 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga
     5761 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     5821 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     5881 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     5941 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     6001 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca
     6061 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     6121 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     6181 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     6241 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     6301 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag
     6361 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     6421 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     6481 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     6541 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     6601 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     6661 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     6721 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     6781 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     6841 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     6901 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc
     6961 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     7021 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     7081 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     7141 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
     7201 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     7261 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     7321 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     7381 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     7441 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     7501 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     7561 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     7621 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     7681 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     7741 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     7801 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     7861 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     7921 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     7981 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     8041 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     8101 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     8161 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     8221 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     8281 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     8341 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     8401 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     8461 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c
//
