LOCUS       Exported                7003 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expression of (-30)GFP-9xGGS-Cre-6xHis in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    pET-(-30)GFP-9xGGS-Cre-6xHis
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7003)
  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 7003)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7003
                     /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             881..1900
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="NLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLS
                     VCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPR
                     PSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLG
                     IAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERW
                     ISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRY
                     LAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLEDG
                     "
     CDS             1913..1930
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1949..1993
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2031..2049)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2045..2092
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2129..2584
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2216..2235)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2426..2447
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2699..3559)
                     /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     3322..3341
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3560..3652)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3733..4321
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4222..4241
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4475..4492
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4507..4649
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4575..4594)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4735..4757
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4751..4942)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5514..6596)
                     /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     6558..6577
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6597..6674)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6880..6899
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6987..3
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6987..2
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atgggtgcta gcaaaggtga agagctgttt gacggtgtag taccgatctt
      121 agtggaatta gacggcgacg tgaacggtca cgaatttagc gtgcgcggcg agggcgaagg
      181 tgacgctacc gagggtgaat tgaccctgaa gtttatttgc acaacaggcg aattacccgt
      241 tccgtggccc accttagtga ccaccctgac ctatggcgtt cagtgcttca gtgattaccc
      301 agatcatatg gatcaacacg attttttcaa atcagccatg cctgaaggat atgttcaaga
      361 gcgtacaatc agcttcaagg acgatggcac ctataaaacg cgtgcggaag tgaaatttga
      421 aggcgacaca ttagtaaacc gtatcgaact gaaaggtatc gacttcaaag aagacggcaa
      481 cattttaggc cataagctgg aatataactt taattctcat gacgtgtata ttacggccga
      541 taaacaggaa aacggtatca aggcagaatt tgaaattcgc cataacgtgg aggacggcag
      601 cgttcaatta gcggatcatt atcaacaaaa cacgccgatt ggtgatgggc ctgtactgtt
      661 acctgacgat cactacctga gcacggagtc agccctgagc aaagatccga acgaagaccg
      721 cgatcacatg gttctgttag aattcgtgac cgctgcaggc attgatcatg gaatggacga
      781 gctgtacaag accggtggta gcggtggttc tggtggttct ggtggtagcg gcggtagcgg
      841 tggtagcggt ggtagcggtg gcagcggcgg taccgcgagc aatttactga ccgtacacca
      901 aaatttgcct gcattgccgg tcgatgcaac gagtgatgag gttcgcaaga acctgatgga
      961 catgttcagg gatcgccagg cgttttctga gcatacctgg aaaatgcttc tgtccgtttg
     1021 ccggtcgtgg gcggcatggt gcaagttgaa taaccggaaa tggtttcccg cagaacctga
     1081 agatgttcgc gattatcttc tatatcttca ggcgcgcggt ctggcagtaa aaactatcca
     1141 gcaacatttg ggccagctaa acatgcttca tcgtcggtcc gggctgccac gaccaagtga
     1201 cagcaatgct gtttcactgg ttatgcggcg tatccgaaaa gaaaacgttg atgccggtga
     1261 acgtgcaaaa caggctctag cgttcgaacg cactgatttc gaccaggttc gttcactcat
     1321 ggaaaatagc gatcgctgcc aggatatacg taatctggca tttctgggga ttgcttataa
     1381 caccctgtta cgtatagccg aaattgccag gatcagggtt aaagatatct cacgtactga
     1441 cggtgggaga atgttaatcc atattggcag aacgaaaacg ctggttagca ccgcaggtgt
     1501 agagaaggca cttagcctgg gggtaactaa actggtcgag cgatggattt ccgtctctgg
     1561 tgtagctgat gatccgaata actacctgtt ttgccgggtc agaaaaaatg gtgttgccgc
     1621 gccatctgcc accagccagc tatcaactcg cgccctggaa gggatttttg aagcaactca
     1681 tcgattgatt tacggcgcta aggatgactc tggtcagaga tacctggcct ggtctggaca
     1741 cagtgcccgt gtcggagccg cgcgagatat ggcccgcgct ggagtttcaa taccggagat
     1801 catgcaagct ggtggctgga ccaatgtaaa tattgtcatg aactatatcc gtaacctgga
     1861 tagtgaaaca ggggcaatgg tgcgcctgct ggaagatggc gacggcggat cccatcacca
     1921 ccaccatcac taatgactcg agtctggtaa agaaaccgct gctgcgaaat ttgaacgcca
     1981 gcacatggac tcgtctacta gcgcagctta attaacctag gctgctgcca ccgctgagca
     2041 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     2101 aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     2161 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     2221 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     2281 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     2341 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     2401 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     2461 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     2521 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     2581 atttctggcg gcacgatggc atgagattat caaaaaggat cttcacctag atccttttaa
     2641 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     2701 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     2761 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     2821 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     2881 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     2941 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     3001 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     3061 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     3121 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     3181 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     3241 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     3301 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     3361 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     3421 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     3481 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     3541 ggaaatgttg aatactcata ctcttccttt ttcaatcatg attgaagcat ttatcagggt
     3601 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggtcat
     3661 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     3721 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3781 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     3841 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     3901 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3961 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     4021 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     4081 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     4141 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     4201 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     4261 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     4321 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     4381 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     4441 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     4501 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatata
     4561 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     4621 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     4681 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     4741 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     4801 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     4861 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     4921 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
     4981 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
     5041 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga
     5101 gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg
     5161 gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc
     5221 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg
     5281 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct
     5341 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc
     5401 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     5461 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     5521 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5581 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     5641 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     5701 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     5761 ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg caccaacgcg cagcccggac
     5821 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     5881 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     5941 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     6001 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     6061 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     6121 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     6181 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     6241 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     6301 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     6361 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     6421 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     6481 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     6541 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     6601 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     6661 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     6721 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     6781 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     6841 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     6901 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     6961 cgagatcgat ctcgatcccg cgaaattaat acgactcact ata
//
