LOCUS       Exported                6144 bp ds-DNA     circular SYN 12-AUG-2020
DEFINITION  His6-tev-Hs.CALM1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6144)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Aug 19, 2020 from SnapGene Viewer 5.0.8
            https://www.snapgene.com
COMMENT     Entry Clone:   R701-E65, 3158 bps DNA circular (attL1, attL2 sites)
            Destination Vector:   pDest527, 8266 bps DNA circular (attR1, attR2 
            sites)
            Recombination using LR clonase to produce Expression Clone
FEATURES             Location/Qualifiers
     source          1..6144
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        2..20
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    21..45
                     /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)."
     RBS             60..82
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             90..626
                     /codon_start=1
                     /label=His6-tev-Hs.CALM1(2-148)
                     /translation="MRSGSHHHHHHRSDITSLYKKVGENLYFQGADQLTEEQIAEFKEA
                     FSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTIDFPEFLTMMAR
                     KMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGD
                     GQVNYEEFVQMMTAK"
     CDS             105..122
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    135..159
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             159..626
                     /codon_start=1
                     /gene="tev-Hs.CALM1"
                     /label=tev-Hs.CALM1(2-148)
                     /translation="ENLYFQGADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     CDS             159..179
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     protein_bind    complement(625..649)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             748..765
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      931..1017
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1109..1136
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      1204..1251
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1288..1743
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(1858..2718)
                     /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(2719..2811)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2892..3480
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    3666..3808
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(3910..4101)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4673..5755)
                     /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(5756..5833)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt
       61 ttgtttaact ttaagaagga gatatacata tgagatctgg atcccaccat caccatcacc
      121 atagatctga tatcacaagt ttgtacaaaa aagttggcga aaacctgtac ttccaaggcg
      181 ctgatcagct gaccgaagaa cagattgctg aattcaagga agccttctcc ctatttgata
      241 aagatggcga tggcaccatc acaacaaagg aacttggaac tgtcatgagg tcactgggtc
      301 agaacccaac agaagctgaa ttgcaggata tgatcaatga agtggatgct gatggtaatg
      361 gcaccattga cttccccgaa tttttgacca tgatggctag aaaaatgaaa gatacagata
      421 gtgaagaaga aatccgtgag gcattccgag tctttgacaa ggatggcaat ggttatatca
      481 gtgcagcaga actacgtcac gtcatgacaa acttaggaga aaaactaaca gatgaagaag
      541 tagatgaaat gatcagagaa gcagatattg atggagacgg acaagtcaac tatgaagaat
      601 tcgtacagat gatgactgca aaataatcaa ctttcttgta caaagtggtg atatcgctag
      661 ctagatagct agaagcttgc ggccgcacag ctgtatacac gtgcaagcca gccagaactc
      721 gctcctgaag acccagagga tctcgagcac caccaccacc accactaatg ttaattaagt
      781 tgggcgttcc taggctgata aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac
      841 ctgaccccat gccgaactca gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc
      901 cccatgcgag agtagggaac tgccaggcat caaataaaac gaaaggctca gtcgaaagac
      961 tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg
     1021 ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg
     1081 ccataaactg ccaggcatca aattaagcag aaggccatcc tgacggatgg cctttttgcg
     1141 tttctacaaa ctcttttgtt tatttttcta aatacattca aatatgtatc cgctgagcaa
     1201 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     1261 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     1321 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     1381 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     1441 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     1501 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     1561 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     1621 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     1681 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     1741 tttctggcgg cacgatggca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     1801 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     1861 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     1921 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     1981 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     2041 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     2101 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     2161 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     2221 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     2281 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     2341 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     2401 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     2461 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     2521 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     2581 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     2641 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     2701 gaaatgttga atactcatac tcttcctttt tcaatcatga ttgaagcatt tatcagggtt
     2761 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggtcatg
     2821 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     2881 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     2941 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     3001 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     3061 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     3121 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     3181 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     3241 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     3301 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     3361 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     3421 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     3481 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     3541 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     3601 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     3661 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatat
     3721 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     3781 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     3841 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     3901 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     3961 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     4021 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     4081 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
     4141 aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc
     4201 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
     4261 aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg
     4321 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
     4381 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
     4441 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
     4501 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgct
     4561 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     4621 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     4681 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4741 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     4801 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     4861 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     4921 tgtcttcggt atcgtcgtat cccactaccg agatgtccgc accaacgcgc agcccggact
     4981 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     5041 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     5101 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     5161 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     5221 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     5281 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     5341 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     5401 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     5461 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     5521 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     5581 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     5641 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     5701 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     5761 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     5821 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     5881 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     5941 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     6001 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     6061 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
     6121 gagatcgatc tcgatcccgc gaaa
//
