LOCUS       Exported                5916 bp ds-DNA     circular SYN 12-AUG-2020
DEFINITION  His6-tev-Hs.CALM1(76-148).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5916)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Aug 19, 2020 from SnapGene Viewer 5.0.8
            https://www.snapgene.com
COMMENT     Entry Clone:   R701-E72, 2930 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..5916
                     /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..398
                     /codon_start=1
                     /label=His6-tev-Hs.CALM1(76-148)
                     /translation="MRSGSHHHHHHRSDITSLYKKVGENLYFQGMKDTDSEEEIREAFR
                     VFDKDGNGYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTA"
     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..398
                     /codon_start=1
                     /gene="tev-Hs.CALM1(76-148)"
                     /label=tev-Hs.CALM1(76-148)
                     /translation="ENLYFQGMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLG
                     EKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTA"
     CDS             159..179
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     protein_bind    complement(397..421)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             520..537
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      703..789
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      881..908
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      976..1023
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1060..1515
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(1630..2490)
                     /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(2491..2583)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2664..3252
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    3438..3580
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(3682..3873)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4445..5527)
                     /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(5528..5605)
                     /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 ttccaaggca
      181 tgaaagatac agatagtgaa gaagaaatcc gtgaggcatt ccgagtcttt gacaaggatg
      241 gcaatggtta tatcagtgca gcagaactac gtcacgtcat gacaaactta ggagaaaaac
      301 taacagatga agaagtagat gaaatgatca gagaagcaga tattgatgga gacggacaag
      361 tcaactatga agaattcgta cagatgatga ctgcataatc aactttcttg tacaaagtgg
      421 tgatatcgct agctagatag ctagaagctt gcggccgcac agctgtatac acgtgcaagc
      481 cagccagaac tcgctcctga agacccagag gatctcgagc accaccacca ccaccactaa
      541 tgttaattaa gttgggcgtt cctaggctga taaaacagaa tttgcctggc ggcagtagcg
      601 cggtggtccc acctgacccc atgccgaact cagaagtgaa acgccgtagc gccgatggta
      661 gtgtggggtc tccccatgcg agagtaggga actgccaggc atcaaataaa acgaaaggct
      721 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
      781 aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg
      841 gcaggacgcc cgccataaac tgccaggcat caaattaagc agaaggccat cctgacggat
      901 ggcctttttg cgtttctaca aactcttttg tttatttttc taaatacatt caaatatgta
      961 tccgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     1021 ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat
     1081 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     1141 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     1201 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     1261 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     1321 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     1381 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     1441 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     1501 taacgtttac aatttctggc ggcacgatgg catgagatta tcaaaaagga tcttcaccta
     1561 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     1621 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     1681 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     1741 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     1801 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     1861 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     1921 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     1981 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     2041 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     2101 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     2161 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     2221 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     2281 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     2341 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     2401 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     2461 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatcat gattgaagca
     2521 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     2581 aaataggtca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     2641 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     2701 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     2761 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     2821 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     2881 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     2941 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     3001 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     3061 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     3121 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     3181 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     3241 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     3301 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     3361 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     3421 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     3481 caccgcatat atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     3541 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     3601 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     3661 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     3721 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     3781 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     3841 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     3901 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     3961 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     4021 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     4081 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     4141 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     4201 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     4261 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     4321 cacgatcatg ctagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     4381 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     4441 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     4501 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     4561 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     4621 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     4681 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc
     4741 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     4801 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     4861 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     4921 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     4981 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     5041 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     5101 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     5161 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     5221 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     5281 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     5341 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     5401 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     5461 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     5521 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     5581 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     5641 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     5701 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     5761 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     5821 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     5881 gcgtagagga tcgagatcga tctcgatccc gcgaaa
//
