LOCUS       Exported                5896 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expression of 6xHis-(+36)GFP in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    pET-6xHis-(pos36)GFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5896)
  AUTHORS   Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, Maeder
            ML, Joung JK, Chen ZY, Liu DR
  TITLE     Cationic lipid-mediated delivery of proteins enables efficient 
            protein-based genome editing in vitro and in vivo.
  JOURNAL   Nat Biotechnol. 2015 Jan;33(1):73-80. doi: 10.1038/nbt.3081. Epub 
            2014 Oct 30.
  PUBMED    25357182
REFERENCE   2  (bases 1 to 5896)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5896
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    3..27
                     /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             42..64
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             77..94
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             842..886
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(924..942)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      938..985
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1022..1477
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1109..1128)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1319..1340
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1592..2452)
                     /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"
     primer_bind     2215..2234
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2453..2545)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2626..3214
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3115..3134
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3368..3385
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3400..3542
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3468..3487)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3628..3650
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3644..3835)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4407..5489)
                     /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"
     primer_bind     5451..5470
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5490..5567)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5773..5792
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5880..3
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5880..2
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atgggtcatc accaccacca tcacggtggc gctagcaaag gtgaacgtct
      121 gtttcgtggt aaagtaccga tcttagtgga attaaagggc gacgtgaacg gtcataaatt
      181 tagcgtgcgc ggcaaaggca aaggtgacgc tacccgtggt aaattgaccc tgaagtttat
      241 ttgcacaaca ggcaaattac ccgttccgtg gcccacctta gtgaccaccc tgacctatgg
      301 cgttcagtgc ttcagtcgtt accctaaaca tatgaaacgt cacgattttt tcaaatcagc
      361 catgcctaaa ggatatgttc aagagcgtac aatcagcttc aagaaggatg gcaaatataa
      421 aacgcgtgcg gaagtgaaat ttgaaggccg cacattagta aatcgtatca aactgaaagg
      481 tcgtgacttc aaagaaaaag gcaacatttt aggccataaa ctgcgttata actttaattc
      541 tcataaggtg tatattacgg ccgataaacg caagaatggt atcaaggcaa aattcaaaat
      601 tcgccataac gtgaaagacg gcagcgttca attagcggat cattatcaac aaaacacgcc
      661 gattggtcgc gggcctgtac tgttacctcg caaccactac ctgagcaccc gttctaaact
      721 gagcaaagat ccgaaagaaa aacgcgatca catggttctg ttagaattcg tgaccgctgc
      781 aggcattaag cacggacgcg acgaacgcta caagtaataa tgaggtaccc tcgagtctgg
      841 taaagaaacc gctgctgcga aatttgaacg ccagcacatg gactcgtcta ctagcgcagc
      901 ttaattaacc taggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
      961 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg
     1021 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     1081 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     1141 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     1201 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     1261 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     1321 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     1381 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     1441 aacgcgaatt ttaacaaaat attaacgttt acaatttctg gcggcacgat ggcatgagat
     1501 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     1561 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     1621 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     1681 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     1741 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     1801 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     1861 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     1921 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     1981 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     2041 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     2101 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     2161 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     2221 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     2281 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     2341 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     2401 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     2461 tttttcaatc atgattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     2521 gaatgtattt agaaaaataa acaaataggt catgaccaaa atcccttaac gtgagttttc
     2581 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     2641 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2701 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     2761 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     2821 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     2881 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     2941 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3001 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3061 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3121 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3181 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3241 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3301 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3361 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     3421 tacgcatctg tgcggtattt cacaccgcat atatggtgca ctctcagtac aatctgctct
     3481 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     3541 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     3601 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     3661 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     3721 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     3781 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     3841 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     3901 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     3961 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     4021 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     4081 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     4141 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     4201 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     4261 gccgggtcct caacgacagg agcacgatca tgctagtcat gccccgcgcc caccggaagg
     4321 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     4381 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4441 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     4501 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     4561 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     4621 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     4681 accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     4741 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     4801 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     4861 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     4921 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     4981 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     5041 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     5101 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     5161 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     5221 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     5281 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     5341 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     5401 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     5461 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     5521 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     5581 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     5641 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     5701 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     5761 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     5821 gatgccggcc acgatgcgtc cggcgtagag gatcgagatc gatctcgatc ccgcgaaatt
     5881 aatacgactc actata
//
