LOCUS       Exported                5893 bp ds-DNA     circular SYN 12-MAY-2023
DEFINITION  Expression of 6xHis-(-30)GFP in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    pET-6xHis-(-30)GFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5893)
  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 5893)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2023 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5893
                     /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..3539
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3465..3484)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3625..3647
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3641..3832)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4404..5486)
                     /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     5448..5467
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5487..5564)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5770..5789
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5877..3
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5877..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 gtgaagagct
      121 gtttgacggt gtagtaccga tcttagtgga attagacggc gacgtgaacg gtcacgaatt
      181 tagcgtgcgc ggcgagggcg aaggtgacgc taccgagggt gaattgaccc tgaagtttat
      241 ttgcacaaca ggcgaattac ccgttccgtg gcccacctta gtgaccaccc tgacctatgg
      301 cgttcagtgc ttcagtgatt acccagatca tatggatcaa cacgattttt tcaaatcagc
      361 catgcctgaa ggatatgttc aagagcgtac aatcagcttc aaggacgatg gcacctataa
      421 aacgcgtgcg gaagtgaaat ttgaaggcga cacattagta aaccgtatcg aactgaaagg
      481 tatcgacttc aaagaagacg gcaacatttt aggccataag ctggaatata actttaattc
      541 tcatgacgtg tatattacgg ccgataaaca ggaaaacggt atcaaggcag aatttgaaat
      601 tcgccataac gtggaggacg gcagcgttca attagcggat cattatcaac aaaacacgcc
      661 gattggtgat gggcctgtac tgttacctga cgatcactac ctgagcacgg agtcagccct
      721 gagcaaagat ccgaacgaag accgcgatca catggttctg ttagaattcg tgaccgctgc
      781 aggcattgat catggaatgg acgagctgta 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 cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     3481 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     3541 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     3601 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     3661 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     3721 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     3781 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     3841 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     3901 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     3961 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     4021 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     4081 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     4141 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     4201 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     4261 gggtcctcaa cgacaggagc acgatcatgc tagtcatgcc ccgcgcccac cggaaggagc
     4321 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     4381 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4441 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     4501 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     4561 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     4621 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     4681 gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     4741 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     4801 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     4861 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     4921 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     4981 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     5041 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     5101 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     5161 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     5221 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     5281 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     5341 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     5401 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     5461 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     5521 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     5581 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     5641 cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg
     5701 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc
     5761 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
     5821 gccggccacg atgcgtccgg cgtagaggat cgagatcgat ctcgatcccg cgaaattaat
     5881 acgactcact ata
//
