LOCUS       Exported                6817 bp ds-DNA     circular SYN 17-AUG-2020
DEFINITION  T7p> RBS-His6-Halotag-tev-RAF1-RBDwitt.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6817)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Aug 19, 2020 from SnapGene Viewer 5.0.8
            https://www.snapgene.com
COMMENT     Entry Clone:   C453-E26, 2440 bps DNA circular (attL4, attL5 sites)
            Entry Clone:   C514-E54, 3673 bps DNA circular (attR5, attR1 sites)
            Entry Clone:   R702-E66, 2951 bps DNA circular (attL1, attL2 sites)
            Destination Vector:   pDest-892, 6988 bps DNA circular (attR4, attR2
            sites)
            Recombination using LR clonase to produce Expression Clone
FEATURES             Location/Qualifiers
     source          1..6817
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             96..113
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      279..365
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      457..484
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      552..599
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      636..1091
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(1206..2066)
                     /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(2067..2159)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2240..2828
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    3014..3156
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(3258..3449)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4021..5103)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(5104..5181)
                     /gene="lacI"
                     /label=lacI promoter
     protein_bind    5282..5302
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        5435..5453
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5454..5478
                     /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)."
     protein_bind    complement(5494..5514)
                     /gene="mutant version of attB"
                     /label=attB5
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     CDS             5544..6791
                     /codon_start=1
                     /label=His6-Halotag-tev-Hs.RAF1(52-131)
                     /translation="MGHHHHHHMAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLF
                     LHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALG
                     LEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTT
                     DVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGE
                     PANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNL
                     LQEDNPDLIGSEIARWLSTLEISGEPTTEDLYFQSDNAIATTLYKKVGENLYFQSKTSN
                     TIRVFLPNKQRTVVNVRNGMSLHDCLMKALKVRGLQPECCAVFRLLHEHKGKKARLDWN
                     TDAASLIGEELQVDFL"
     CDS             5550..5567
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5568..6458
                     /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             6471..6491
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     protein_bind    6507..6531
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             6531..6791
                     /codon_start=1
                     /label=tev-Hs.RAF1(52-131)
                     /translation="ENLYFQSKTSNTIRVFLPNKQRTVVNVRNGMSLHDCLMKALKVRG
                     LQPECCAVFRLLHEHKGKKARLDWNTDAASLIGEELQVDFL"
     CDS             6531..6551
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     protein_bind    complement(6790..6814)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 atcgctagct agatagctag aagcttgcgg ccgcacagct gtatacacgt gcaagccagc
       61 cagaactcgc tcctgaagac ccagaggatc tcgagcacca ccaccaccac cactaatgtt
      121 aattaagttg ggcgttccta ggctgataaa acagaatttg cctggcggca gtagcgcggt
      181 ggtcccacct gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt
      241 ggggtctccc catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt
      301 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga
      361 caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag
      421 gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg acggatggcc
      481 tttttgcgtt tctacaaact cttttgttta tttttctaaa tacattcaaa tatgtatccg
      541 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
      601 tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag
      661 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
      721 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
      781 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
      841 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
      901 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
      961 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     1021 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     1081 gtttacaatt tctggcggca cgatggcatg agattatcaa aaaggatctt cacctagatc
     1141 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     1201 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     1261 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     1321 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     1381 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     1441 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     1501 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     1561 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     1621 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     1681 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     1741 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     1801 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     1861 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     1921 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     1981 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     2041 gcgacacgga aatgttgaat actcatactc ttcctttttc aatcatgatt gaagcattta
     2101 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     2161 aggtcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     2221 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     2281 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     2341 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     2401 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     2461 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     2521 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     2581 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     2641 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     2701 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     2761 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     2821 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     2881 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     2941 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     3001 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     3061 gcatatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     3121 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     3181 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     3241 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     3301 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     3361 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     3421 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     3481 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     3541 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     3601 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     3661 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     3721 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     3781 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     3841 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     3901 atcatgctag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     3961 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     4021 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     4081 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     4141 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     4201 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     4261 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atgtccgcac caacgcgcag
     4321 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     4381 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     4441 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     4501 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     4561 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
     4621 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     4681 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     4741 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     4801 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     4861 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
     4921 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     4981 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     5041 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     5101 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     5161 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta
     5221 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgatc
     5281 acaactttgt atagaaaagt tggctggcga gcccgatctt ccccatcggt gatgtcggcg
     5341 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     5401 tagaggatcg agatcgatct cgatcccgcg aaattaatac gactcactat aggggaattg
     5461 tgagcggata acaattcccc tctagaaata agccaacttt tgtatacaaa gtggtcattt
     5521 aactttaaga aggagatata catatgggcc accatcacca tcaccatatg gcagaaatcg
     5581 gtactggctt tccattcgac ccccattatg tggaagtcct gggcgagcgc atgcactacg
     5641 tcgatgttgg tccgcgcgat ggcacccctg tgctgttcct gcacggtaac ccgacctcct
     5701 cctacgtgtg gcgcaacatc atcccgcatg ttgcaccgac ccatcgctgc attgctccag
     5761 acctgatcgg tatgggcaaa tccgacaaac cagacctggg ttatttcttc gacgaccacg
     5821 tccgcttcat ggatgccttc atcgaagccc tgggtctgga agaggtcgtc ctggtcattc
     5881 acgactgggg ctccgctctg ggtttccact gggccaagcg caatccagag cgcgtcaaag
     5941 gtattgcatt tatggagttc atccgcccta tcccgacctg ggacgaatgg ccagaatttg
     6001 cccgcgagac cttccaggcc ttccgcacca ccgacgtcgg ccgcaagctg atcatcgatc
     6061 agaacgtttt tatcgagggt acgctgccga tgggtgtcgt ccgcccgctg actgaagtcg
     6121 agatggacca ttaccgcgag ccgttcctga atcctgttga ccgcgagcca ctgtggcgct
     6181 tcccaaacga gctgccaatc gccggtgagc cagcgaacat cgtcgcgctg gtcgaagaat
     6241 acatggactg gctgcaccag tcccctgtcc cgaagctgct gttctggggc accccaggcg
     6301 ttctgatccc accggccgaa gccgctcgcc tggccaaaag cctgcctaac tgcaaggctg
     6361 tggacatcgg cccgggtctg aatctgctgc aagaagacaa cccggacctg atcggcagcg
     6421 agatcgcgcg ctggctgtcg acgctcgaga tttccggcga gccaaccact gaggatctgt
     6481 actttcagag cgataacgcg atcgccacaa ctttgtacaa aaaagttggc gagaacctgt
     6541 actttcagtc caagaccagc aacacgatcc gcgtgttcct gccgaataag caacgtaccg
     6601 tcgttaatgt gcgcaacggt atgagcctgc atgactgcct gatgaaagca ctgaaagtgc
     6661 gtggtctgca gccggagtgt tgtgcggtct ttcgtctgct gcacgagcat aagggcaaaa
     6721 aagcccgtct ggactggaat accgatgcag cgagcctgat tggtgaagaa ctgcaagtcg
     6781 acttcttgta atcaactttc ttgtacaaag ttgtgat
//
