LOCUS       Exported                7790 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses IUP pathway genes (ChK, IPK, idi) under inducible 
            promoter. aTC inducible. Kan. resist..
ACCESSION   .
VERSION     .
KEYWORDS    pTET-IUPi
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7790)
  AUTHORS   Chatzivasileiou AO, Ward V, Edgar SM, Stephanopoulos G
  TITLE     Two-step pathway for isoprenoid synthesis.
  JOURNAL   Proc Natl Acad Sci U S A. 2018 Dec 24. pii: 1812935116. doi: 
            10.1073/pnas.1812935116.
  PUBMED    30584096
REFERENCE   2  (bases 1 to 7790)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7790
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     154..171
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    186..325
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(251..270)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     411..433
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(427..618)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1427..2050)
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     1958..1977
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        2069..2124
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    2105..2123
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             3932..3940
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     RBS             4947..4955
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             5550..5567
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5620..5638)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5634..5681
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5718..6173
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5805..5824)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6015..6036
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6265..7080)
                     /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     6988..7007
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7072..7091
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      7202..7790
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7691..7710
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
       61 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
      121 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
      181 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
      241 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
      301 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
      361 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
      421 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
      481 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
      541 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
      601 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
      661 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
      721 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
      781 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
      841 ccagcagcat cctgcgatgc agatccggaa cataattgtg cagggcgctg acttccgcgt
      901 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
      961 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     1021 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     1081 ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     1141 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     1201 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     1261 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     1321 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     1381 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgcttaa gacccacttt
     1441 cacatttaag ttgtttttct aatccgcata tgatcaattc aaggccgaat aagaaggctg
     1501 gctctgcacc ttggtgatca aataattcga tagcttgtcg taataatggc ggcatactat
     1561 cagtagtagg tgtttccctt tcttctttag cgacttgatg ctcttgatct tccaatacgc
     1621 aacctaaagt aaaatgcccc acagcgctga gtgcatataa tgcattctct agtgaaaaac
     1681 cttgttggca taaaaaggct aattgatttt cgagagtttc atactgtttt tctgtaggcc
     1741 gtgtacctaa atgtactttt gctccatcgc gatgacttag taaagcacat ctaaaacttt
     1801 tagcgttatt acgtaaaaaa tcttgccagc tttccccttc taaagggcaa aagtgagtat
     1861 ggtgcctatc taacatctca atggctaagg cgtcgagcaa agcccgctta ttttttacat
     1921 gccaatacaa tgtaggctgc tctacaccta gcttctgggc gagtttacgg gttgttaaac
     1981 cttcgattcc gacctcatta agcagctcta atgcgctgtt aatcacttta cttttatcta
     2041 atctagacat cattaattcc taatttttgt tgacactcta tcgttgatag agttatttta
     2101 ccactcccta tcagtgatag agaaaagaat tcaaaagatc ttttaagaag gagatataca
     2161 tatggtgcag gagtcccgcc ccggctcggt ccggtcgtat tccgtgggct accaggcccg
     2221 gtcgcggtcg tcgtcccagc gccgccattc gctcacgcgg cagcgcagca gccagcggct
     2281 catccggacg atctccatcg agagcgatgt gagcaatatc acggacgatg atgatctgcg
     2341 ggcggtgaat gaaggggtgg ccggggtcca gctcgacgtc tccgagacgg cgaacaaagg
     2401 gccacgccgg gccagtgcca ccgatgtcac cgactcgctg ggctccacgt ccagcgaata
     2461 tatcgagatc cccttcgtga aagagacgct ggacgcgagc ctcccctcgg attacctcaa
     2521 acaagacatc ctgaacctga tccaatccct gaagatctcg aaatggtaca ataacaaaaa
     2581 gatccagccc gtcgcccagg acatgaacct cgtcaaaatc tccggcgcga tgaccaatgc
     2641 gatcttcaag gtggagtacc cgaaactgcc gtccctcctg ctgcggatct atggcccgaa
     2701 tatcgataac atcatcgacc gcgaatatga actccagatc ctcgcgcggc tctcgctgaa
     2761 aaacatcggg ccgtccctgt acggctgctt cgtgaatggg cgcttcgagc agttcctcga
     2821 aaactccaaa acgctgacca aggatgatat ccggaactgg aaaaactcgc aacggatcgc
     2881 ccgccgcatg aaggagctgc atgtgggcgt gcccctcctc tcgtcggagc ggaagaatgg
     2941 gagcgcctgc tggcaaaaaa tcaaccaatg gctccgcacg atcgagaagg tggatcagtg
     3001 ggtcggggac ccgaagaaca tcgagaacag cctcctctgc gaaaattggt ccaaattcat
     3061 ggacatcgtc gatcggtacc acaagtggct gatcagccaa gaacaaggga tcgagcaagt
     3121 caacaaaaat ctgatcttct gccataatga tgcccaatac gggaatctcc tcttcaccgc
     3181 gcccgtcatg aacaccccct ccctgtatac cgcgccgagc tcgacctccc tgacgtccca
     3241 aagcagcagc ctcttcccct cgtccagcaa cgtgatcgtc gatgatatca tcaatccccc
     3301 gaagcaagaa caatcccaag attccaaact cgtggtcatc gatttcgaat acgccggggc
     3361 caatcccgcc gcgtacgatc tcgccaatca cctctcggaa tggatgtacg actataataa
     3421 cgccaaagcc ccgcaccagt gccacgccga ccggtacccc gacaaggagc aagtgctcaa
     3481 cttcctgtat tcgtatgtca gccatctccg cggcggggcc aaagagccca tcgatgaaga
     3541 agtccagcgc ctctataaat cgatcatcca gtggcgcccc acggtgcagc tcttctggtc
     3601 gctgtgggcg atcctgcaaa gcggcaagct ggaaaaaaaa gaagccagca ccgccatcac
     3661 ccgcgaagaa atcgggccca atgggaaaaa gtatatcatc aagacggagc ccgagtcgcc
     3721 cgaagaggac ttcgtcgaaa atgacgacga acccgaagcc ggcgtgtcga tcgatacctt
     3781 cgactacatg gcctacgggc gggacaagat cgcggtgttc tggggggacc tgatcgggct
     3841 gggcatcatc acggaggagg aatgcaagaa cttctcgagc ttcaaattcc tcgacaccag
     3901 ctacctgtaa gtacaagaaa agtcagtagt ctaaggaggt aagcatggaa ctcaatatca
     3961 gcgaaagccg gtcgcgcagc atccggtgca tcgtgaagct cgggggcgcg gccatcacct
     4021 gcaaaaacga actcgaaaag atccatgacg aaaacctcga agtggtggcc tgccaactgc
     4081 ggcaagcgat gctggaaggc tccgccccct ccaaagtcat cggcatggac tggtccaaac
     4141 ggccgggctc ctccgaaatc tcctgcgatg tggacgacat cggcgaccag aaatcgagcg
     4201 aattctcgaa gttcgtggtc gtccacgggg cgggctcgtt cggccatttc caagcgtcgc
     4261 ggtcgggcgt ccataaaggg ggcctggaga agcccatcgt caaagccggg ttcgtcgcga
     4321 cccggatctc ggtcaccaat ctcaatctcg agatcgtccg cgccctggcc cgggaaggca
     4381 tcccgacgat cgggatgagc cccttcagct gcggctggtc cacctccaaa cgggatgtcg
     4441 cgtcggcgga tctcgcgacg gtcgccaaga ccatcgactc gggcttcgtg cccgtgctcc
     4501 atggggacgc ggtcctggac aacatcctgg gctgcacgat cctgtccggc gatgtcatca
     4561 tccggcacct ggccgaccat ctcaagccgg aatacgtcgt gttcctgacc gacgtgctcg
     4621 gggtctatga ccggcccccg agcccgtcgg agccggacgc cgtcctgctc aaggagatcg
     4681 ccgtcgggga ggatggctcc tggaaggtgg tcaaccccct cctggaacat accgacaaaa
     4741 aggtggatta ttccgtcgcg gcccacgata ccacgggggg gatggaaacc aaaatctcgg
     4801 aagccgccat gatcgccaag ctcggcgtgg atgtgtacat cgtgaaagcc gcgaccaccc
     4861 attcgcagcg ggcgctcaat ggcgacctgc gcgactccgt ccccgaagac tggctgggga
     4921 ccatcatccg cttcagcaaa taaaactaag gaggtctata tgcaaacgga acacgtcatt
     4981 ttattgaatg cacagggagt tcccacgggt acgctggaaa agtatgccgc acacacggca
     5041 gacacccgct tacatctcgc gttctccagt tggctgttta atgccaaagg acaattatta
     5101 gttacccgcc gcgcactgag caaaaaagca tggcctggcg tgtggactaa ctcggtttgt
     5161 gggcacccac aactgggaga aagcaacgaa gacgcagtga tccgccgttg ccgttatgag
     5221 cttggcgtgg aaattacgcc tcctgaatct atctatcctg actttcgcta ccgcgccacc
     5281 gatccgagtg gcattgtgga aaatgaagtg tgtccggtat ttgccgcacg caccactagt
     5341 gcgttacaga tcaatgatga tgaagtgatg gattatcaat ggtgtgattt agcagatgta
     5401 ttacacggta ttgatgccac gccgtgggcg ttcagtccgt ggatggtgat gcaggcgaca
     5461 aatcgcgaag ccagaaaacg attatctgca tttacccagc ttaaataacg aattcgagct
     5521 ccgtcgacaa gcttgcggcc gcactcgagc accaccacca ccaccactga gatccggctg
     5581 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
     5641 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     5701 ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     5761 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     5821 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     5881 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     5941 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     6001 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     6061 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     6121 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     6181 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     6241 atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa
     6301 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg
     6361 agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc
     6421 gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag
     6481 tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc
     6541 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac
     6601 caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa
     6661 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac
     6721 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat
     6781 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag
     6841 aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac
     6901 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata
     6961 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc
     7021 atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat
     7081 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc
     7141 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     7201 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     7261 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     7321 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     7381 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     7441 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     7501 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     7561 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     7621 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     7681 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     7741 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
//
