LOCUS       Exported                7721 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  FX cloning E.coli expression vector with T7 promoter and C-terminal 
            3C protease cleavage site, GFP and 10x His tags.
ACCESSION   .
VERSION     .
KEYWORDS    p7XC3GH
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7721)
  AUTHORS   Geertsma ER, Dutzler R
  TITLE     A versatile and efficient high-throughput cloning tool for 
            structural biology.
  JOURNAL   Biochemistry. 2011 Apr 19;50(15):3272-8. Epub 2011 Mar 25.
  PUBMED    21410291
REFERENCE   2  (bases 1 to 7721)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7721
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             6..23
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(76..94)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      90..137
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      174..629
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(261..280)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     471..492
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(722..1537)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1445..1464
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1529..1548
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1659..2247
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2148..2167
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2401..2418
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2433..2575
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2501..2520)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2661..2683
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2677..2868)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3677..4759)
                     /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     4721..4740
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4760..4837)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5043..5062
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5146..5165
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5146..5164
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5165..5189
                     /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             5204..5226
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             complement(5285..5590)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(5387..5406)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(5932..6591)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6506..6525
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6645..6675)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(6700..6824)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(6843..6863)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(6867..6887)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6914..6937
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             6944..7654
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKAN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGTDELYK"
     primer_bind     complement(6971..6999)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     7594..7614
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             7661..7690
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
ORIGIN
        1 tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg
       61 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
      121 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc
      181 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
      241 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
      301 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
      361 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
      421 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
      481 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
      541 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
      601 ttttaacaaa atattaacgt ttacaatttc aggtggcact tttcggggaa atgtgcgcgg
      661 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
      721 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
      781 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
      841 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
      901 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
      961 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     1021 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     1081 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     1141 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     1201 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     1261 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     1321 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     1381 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     1441 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     1501 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     1561 tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac
     1621 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     1681 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     1741 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     1801 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     1861 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     1921 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     1981 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     2041 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     2101 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     2161 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     2221 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     2281 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     2341 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     2401 agcgagtcag tgagcgagga agcggatcag cgcctgatgc ggtattttct ccttacgcat
     2461 ctgtgcggta tttcacaccg catatatggt gcactctcag tacaatctgc tctgatgccg
     2521 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     2581 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     2641 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     2701 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
     2761 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct
     2821 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt
     2881 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac
     2941 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact
     3001 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt
     3061 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa
     3121 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa
     3181 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg
     3241 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt
     3301 cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc
     3361 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
     3421 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
     3481 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
     3541 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
     3601 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     3661 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     3721 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     3781 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     3841 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     3901 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat
     3961 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     4021 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     4081 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     4141 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     4201 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     4261 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     4321 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     4381 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     4441 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     4501 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     4561 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     4621 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     4681 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     4741 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     4801 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     4861 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     4921 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     4981 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     5041 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
     5101 acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta
     5161 taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga
     5221 aggagatata catatgagtt gaagagcgac ctgcagactg gctgtgtata agggagcctg
     5281 acatttatat tccccagaac atcaggttaa tggcgttttt gatgtcattt tcgcggtggc
     5341 tgagatcagc cacttcttcc ccgataacgg agaccggcac actggccata tcggtggtca
     5401 tcatgcgcca gctttcatcc ccgatatgca ccaccgggta aagttcacgg gagactttat
     5461 ctgacagcag acgtgcactg gccaggggga tcaccatccg tcgcccgggc gtgtcaataa
     5521 tatcactctg tacatccaca aacagacgat aacggctctc tcttttatag gtgtaaacct
     5581 taaactgcat ttcaccagcc cctgttctcg tcagcaaaag agccgttcat ttcaataaac
     5641 cgggcgacct cagccatccc ttcctgattt tccgctttcc agcgttcggc acgcagacga
     5701 cgggcttcat tctgcatggt tgtgcttacc agaccggaga tattgacatc atatatgcct
     5761 tgagcaactg atagctgtcg ctgtcaactg tcactgtaat acgctgcttc atagcatacc
     5821 tctttttgac atacttcggg tatacatatc agtatatatt cttataccgc aaaaatcagc
     5881 gcgcaaatac gcatactgtt atctggcttt tagtaagccg gatccacgcg tttacgcccc
     5941 gccctgccac tcatcgcagt actgttgtaa ttcattaagc attctgccga catggaagcc
     6001 atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt
     6061 ataatatttg cccatggtga aaacgggggc gaagaagttg tccatattgg ccacgtttaa
     6121 atcaaaactg gtgaaactca cccagggatt ggctgagacg aaaaacatat tctcaataaa
     6181 ccctttaggg aaataggcca ggttttcacc gtaacacgcc acatcttgcg aatatatgtg
     6241 tagaaactgc cggaaatcgt cgtggtattc actccagagc gatgaaaacg tttcagtttg
     6301 ctcatggaaa acggtgtaac aagggtgaac actatcccat atcaccagct caccgtcttt
     6361 cattgccata cggaattccg gatgagcatt catcaggcgg gcaagaatgt gaataaaggc
     6421 cggataaaac ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa tatccagctg
     6481 aacggtctgg ttataggtac attgagcaac tgactgaaat gcctcaaaat gttctttacg
     6541 atgccattgg gatatatcaa cggtggtata tccagtgatt tttttctcca ttttagcttc
     6601 cttagctcct gaaaatctcg acggatccta actcaaaatc cacacattat acgagccgga
     6661 agcataaagt gtaaagcctg gggtgcctaa tgcggccgcc aatatgactg gatatgttgt
     6721 gttttacagt attatgtagt ctgtttttta tgcaaaatct aatttaatat attgatattt
     6781 atatcatttt acgtttctcg ttcagctttt ttgtacaaac ttgtgatatc gaattcgcta
     6841 gctacctttc tcttcttttt tggatctacc tttctcttct tttttgggga tccactagtt
     6901 cagctcttct gcattagaag ttttgtttca aggtccacaa ttcagcaaag gagaagaact
     6961 tttcactgga gttgtcccaa ttcttgttga attagatggt gatgttaatg ggcacaaatt
     7021 ttctgtcagt ggagagggtg aaggtgatgc tacatacgga aaactcaccc ttaaatttat
     7081 ttgcactact ggaaaactac ctgttccttg gccaacactt gtcactactc tgacctatgg
     7141 tgttcaatgc ttttcccgtt atccggatca catgaaacgg catgactttt tcaagagtgc
     7201 catgcccgaa ggttatgtac aggaacgcac tatatctttc aaagatgacg ggaactacaa
     7261 gacgcgtgct gaagtcaagt ttgaaggtga tacccttgtt aatcgtatcg agttaaaggg
     7321 tattgatttt aaagaagatg gaaacattct cggacacaaa ctagagtaca actataactc
     7381 acacaatgta tacatcacgg cagacaaaca aaagaatgga atcaaagcta acttcaaaat
     7441 tcgccacaac attgaagatg gttccgttca actagcagac cattatcaac aaaatactcc
     7501 aattggcgat ggccctgtcc ttttaccaga caaccattac ctgtcgacac aatctgccct
     7561 ttcgaaagat cccaacgaaa agcgtgacca catggtcctt cttgagtttg taactgctgc
     7621 tgggattaca catggcacgg atgagctcta caaaactagt catcatcatc accatcatca
     7681 tcaccatcat taagtcgacc atctagaaca aaaactcatc c
//
