LOCUS       Exported                5937 bp ds-DNA     circular SYN 14-AUG-2020
DEFINITION  His6-tev-Hs.CALM1(1-80).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5937)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Aug 19, 2020 from SnapGene Viewer 5.0.8
            https://www.snapgene.com
COMMENT     Entry Clone:   R701-E71, 2951 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..5937
                     /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..419
                     /codon_start=1
                     /label=His6-tev-Hs.CALM1(1-80)
                     /translation="MRSGSHHHHHHRSDITSLYKKVGENLYFQGADQLTEEQIAEFKEA
                     FSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTIDFPEFLTMMAR
                     KMKDT"
     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..419
                     /codon_start=1
                     /gene="tev-Hs.CALM1(1-80)"
                     /label=tev-Hs.CALM1(1-80)
                     /translation="ENLYFQGADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGNGTIDFPEFLTMMARKMKDT"
     CDS             159..179
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     protein_bind    complement(418..442)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             541..558
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      724..810
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      902..929
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      997..1044
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1081..1536
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(1651..2511)
                     /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(2512..2604)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2685..3273
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    3459..3601
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(3703..3894)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4466..5548)
                     /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(5549..5626)
                     /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 gatacataat
      421 caactttctt gtacaaagtg gtgatatcgc tagctagata gctagaagct tgcggccgca
      481 cagctgtata cacgtgcaag ccagccagaa ctcgctcctg aagacccaga ggatctcgag
      541 caccaccacc accaccacta atgttaatta agttgggcgt tcctaggctg ataaaacaga
      601 atttgcctgg cggcagtagc gcggtggtcc cacctgaccc catgccgaac tcagaagtga
      661 aacgccgtag cgccgatggt agtgtggggt ctccccatgc gagagtaggg aactgccagg
      721 catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg
      781 tcggtgaacg ctctcctgag taggacaaat ccgccgggag cggatttgaa cgttgcgaag
      841 caacggcccg gagggtggcg ggcaggacgc ccgccataaa ctgccaggca tcaaattaag
      901 cagaaggcca tcctgacgga tggccttttt gcgtttctac aaactctttt gtttattttt
      961 ctaaatacat tcaaatatgt atccgctgag caataactag cataacccct tggggcctct
     1021 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
     1081 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     1141 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     1201 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     1261 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     1321 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     1381 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     1441 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     1501 acgcgaattt taacaaaata ttaacgttta caatttctgg cggcacgatg gcatgagatt
     1561 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     1621 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     1681 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     1741 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     1801 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     1861 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     1921 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     1981 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     2041 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     2101 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     2161 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     2221 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     2281 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     2341 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     2401 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     2461 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     2521 ttttcaatca tgattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     2581 aatgtattta gaaaaataaa caaataggtc atgaccaaaa tcccttaacg tgagttttcg
     2641 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     2701 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     2761 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     2821 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     2881 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     2941 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3001 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3061 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     3121 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     3181 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     3241 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     3301 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     3361 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     3421 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     3481 acgcatctgt gcggtatttc acaccgcata tatggtgcac tctcagtaca atctgctctg
     3541 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     3601 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     3661 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     3721 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     3781 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     3841 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     3901 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     3961 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     4021 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     4081 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     4141 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     4201 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     4261 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     4321 ccgggtcctc aacgacagga gcacgatcat gctagtcatg ccccgcgccc accggaagga
     4381 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     4441 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     4501 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     4561 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     4621 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     4681 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     4741 ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     4801 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     4861 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     4921 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     4981 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     5041 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     5101 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     5161 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     5221 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     5281 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     5341 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     5401 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     5461 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     5521 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     5581 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     5641 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     5701 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     5761 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     5821 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     5881 atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg atctcgatcc cgcgaaa
//
