LOCUS       Exported                8682 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  ABA-inducible split T7 GFP vector using RNAPN d5-19.
ACCESSION   .
VERSION     .
KEYWORDS    pJin-278
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8682)
  AUTHORS   Pu J, Kentala K, Dickinson BC
  TITLE     Multidimensional Control of Cas9 by Evolved RNA Polymerase-Based 
            Biosensors.
  JOURNAL   ACS Chem Biol. 2018 Feb 16;13(2):431-437. doi: 
            10.1021/acschembio.7b00532. Epub 2017 Aug 22.
  PUBMED    28809467
REFERENCE   2  (bases 1 to 8682)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8682
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    465..748
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
     terminator      777..823
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(997..1716)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(1000..1716)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1042..1063)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1391..1410
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1650..1671
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     misc_feature    1717..2303
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1890..1909)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     2119..2136
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     complement(2350..2369)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2351..2369)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2395..2494
                     /label=CMVd1 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields slightly reduced expression 
                     relative to the full-length promoter"
     promoter        5268..5367
                     /label=CMVd1 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields slightly reduced expression 
                     relative to the full-length promoter"
     primer_bind     complement(6902..6920)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    6957..7078
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6968..6987)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7022..7041
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             7477..8271
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MLEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(7531..7550)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     8141..8160
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(8443..349)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8523..8542)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
       61 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
      121 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
      181 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
      241 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
      301 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatttcaag aagatccttt
      361 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
      421 catgagatta tcaaaaagga tcttcaccta gatcctttta tagtccggaa atacaggaac
      481 gcacgctgga tggcccttcg ctgggatggt gaaaccatga aaaatggcag cttcagtgga
      541 ttaagtgggg gtaatgtggc ctgtaccctc tggttgcata ggtattcata cggttaaaat
      601 ttatcaggcg cgattgcggc agtttttcgg gtggtttgtt gccattttta cctgtctgct
      661 gccgtgatcg cgctgaacgc gttttagcgg tgcgtacaat taagggatta tggtaaatcc
      721 acttactgtc tgccctcgta gccatcgaga taaaccgcag tactccggcc acgatgcaaa
      781 aaacccctca agacccgttt agaggcccca aggggttatg ctatcagaag ctttcatttc
      841 agcttcagtt ttcagatgtt tgaaacgatc gaatttttcc agagtttccg gatgagattt
      901 gaacagtcga atcaagatgt cctgaccatg accagcgacg tcagcttcaa ctttagccca
      961 aacatgcaga accagctgcc attcaccttc agcgctttac ttgtacagct cgtccatgcc
     1021 gagagtgatc ccggcggcgg tcacgaactc cagcaggacc atgtgatcgc gcttctcgtt
     1081 ggggtctttg ctcagggcgg actgggtgct caggtagtgg ttgtcgggca gcagcacggg
     1141 gccgtcgccg atgggggtgt tctgctggta gtggtcggcg agctgcacgc tgccgtcctc
     1201 gatgttgtgg cggatcttga agttcacctt gatgccgttc ttctgcttgt cggccatgat
     1261 atagacgttg tggctgttgt agttgtactc cagcttgtgc cccaggatgt tgccgtcctc
     1321 cttgaagtcg atgcccttca gctcgatgcg gttcaccagg gtgtcgccct cgaacttcac
     1381 ctcggcgcgg gtcttgtagt tgccgtcgtc cttgaagaag atggtgcgct cctggacgta
     1441 gccttcgggc atggcggact tgaagaagtc gtgctgcttc atgtggtcgg ggtagcggct
     1501 gaagcactgc acgccgtagg tcagggtggt cacgagggtg ggccagggca cgggcagctt
     1561 gccggtggtg cagatgaact tcagggtcag cttgccgtag gtggcatcgc cctcgccctc
     1621 gccggacacg ctgaacttgt ggccgtttac gtcgccgtcc agctcgacca ggatgggcac
     1681 caccccggtg aacagctcct cgcccttgct caccatggtt gtggccatat tatcatcgtg
     1741 tttttcaaag gaaaaccacg tccccgtggt tcggggggcc tagacgtttt tttaacctcg
     1801 actaaacaca tgtaaagcat gtgtaccgag gccccagatc agatcccata caatggggta
     1861 ccttctgggc atccttcagc cccttgttga atacgcttga ggagagccat ttgactcttt
     1921 ccacaactat ccaactcaca acgtggcact ggggttgtgc cgcctttgca ggtgtatctt
     1981 atacacgtgg cttttggccg cagaggcacc tgtcgccagg tggggggttc cgctgcctgc
     2041 aaagggtcgc tacagacgtt gtttgtcttc aagaagcttc cagaggaact gcttccttca
     2101 cgacattcaa cagaccttgc attcctttgg cgagagggga aagaccccta ggaatgctcg
     2161 tcaagaagac agggccaggt ttccgggccc tcacattgcc aaaagacggc aatatggtgg
     2221 aaaataacat atagacaaac gcacaccggc cttattccaa gcggcttcgg ccagtaacgt
     2281 tagggggggg ggagggagag gggcggatcc gggccctcta gatgcggccg catgcataag
     2341 cttgggtctc cctatagtga gtcgtattac gtccggcgta gaggatcgag atctccaaaa
     2401 tcaacgggac tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa tgggcggtag
     2461 gcgtgtacgg tgggaggtct atataagcat agctggttta gtgaaccgtc agatcactag
     2521 aagcttacgc taccaccatg acccaggacg agtttaccca gctgtcacag tcaatcgccg
     2581 agtttcacac ctatcagctg gggaacggaa gatgtagtag tctgctggcc cagagaatcc
     2641 acgctccccc tgaaaccgtg tggagcgtgg tccggagatt cgatagacca caaatctaca
     2701 agcacttcat caagagctgc aacgtgtccg aagatttcga gatgcgcgtc ggatgtacac
     2761 gagacgtgaa cgtcatctct ggcctgcccg caaatactag tcgagagcgg ctggacctgc
     2821 tggacgatga caggcgcgtg actgggttct ccattaccgg cggggaacac agactgagga
     2881 actacaagtc tgtgaccaca gtccataggt ttgagaaaga ggaagaggaa gagcgcattt
     2941 ggaccgtggt cctggaaagc tacgtggtcg atgtgcccga gggcaatagc gaagaggaca
     3001 caaggctgtt tgccgatact gtgattcggc tgaacctgca gaaactggca agcattactg
     3061 aggctatgaa cacgtcgggt ggaagcggta aagcatttat gcaagttgtc gaggctgaca
     3121 tgctctctaa gggtctactc ggtggcgagg cgtggtcttc gtggcataag gaagactcta
     3181 ttcatgtagg agtacgctgc atcgagatgc tcattgagtc aaccggaatg gttagcttac
     3241 accgccaaaa tgctggcgta gtaggtcaag actctgagac tatcgaactc gcacctgaat
     3301 acgctgaggc tatcgcaacc cgtgcaggtg cgctggctgg catctctccg atgttccaac
     3361 cttgcgtagt tcctcctaag ccgtggactg gcattactgg tggtggctat tgggctaacg
     3421 gtcgtcgtcc tctggcgctg gtgcgtactc acagtaagaa agcactgatg cgctacgaag
     3481 acgtttacat gcctgaggtg tacaaagcga ttaacattgc gcaaaacacc gcatggaaaa
     3541 tcaacaagaa agtcctagcg gtcgccaacg taatcaccaa gtggaagcat tgtccggtcg
     3601 aggacatccc tgcgattgag cgtgaagaac tcccgatgaa accggaagac atcgacatga
     3661 atcctgaggc tctcaccgcg tggaaacgtg ctgccgctgc tgtgtaccgc aaggacaagg
     3721 ctcgcaagtc tcgccgtatc agccttgagt tcatgcttga gcaagccaat aagtttgcta
     3781 accataaggc catctggttc ccttacaaca tggactggcg cggtcgtgtt tacgctgtgt
     3841 caatgttcaa cccgcaaggt aacgatatga ccaaaggact gcttacgctg gcgaaaggta
     3901 aaccaatcgg taaggaaggt tactactggc tgaaaatcca cggtgcaaac tgtgcgggtg
     3961 tcgataaggt tccgttccct gagcgcatca agttcattga ggaaaaccac gagaacatca
     4021 tggcttgcgc taagtctcca ctggagaaca cttggtgggc tgagcaagat tctccgttct
     4081 gcttccttgc gttctgcttt gagtacgctg gggtacagca ccacggcctg agctataact
     4141 gctcccttcc gctggcgttt gacgggtctt gctctggcat ccagcacttc tccgcgatgc
     4201 tccgagatga ggtaggtggt cgcgcggtta acttgcttcc tagtgaaacc gttcaggaca
     4261 tctacgggat tgttgctaag aaagtcaacg agattctaca agcagacgca atcaatggga
     4321 ccgataacga agtagttacc gtgaccgatg agaacactgg tgaaatctct gagaaagtca
     4381 agctgggcac taaggcactg gctggtcaat ggctggctta cggtgttact cgcagtgtga
     4441 ctaagcgttc agtcatgacg ctggcttacg ggtccaaaga gttcggcttc cgtcaacaag
     4501 tgctggaaga taccattcag ccagctattg attccggcaa gggtctgatg ttcactcagc
     4561 cgaatcaggc tgctggatac atggctaagc tgatttggga atctgtgagc gtgacggtgg
     4621 tagctgcggt tgaagcaatg aactggctta agtctgctgc taagctgctg gctgctgagg
     4681 tcaaagataa gaagactgga gagattcttc gcaagcgttg cgctgtgcat tgggtaactc
     4741 ctgatggttt ccctgtgtgg caggaataca agaagcctat tcagacgcgc ttgaacctga
     4801 tgttcctcgg tcagttccgc ttacagccta ccattaacac caacaaagat agcgagattg
     4861 atgcacacaa acaggagtct ggtatcgctc ctaactttgt acacagccaa gacggtagcc
     4921 accttcgtaa gactgtagtg tgggcacacg agaagtacgg aatcgaatct tttgcactga
     4981 ttcacgactc cttcggtacc attccggctg acgctgcgaa cctgttcaaa gcagtgcgcg
     5041 aaactatggt tgacacatat gagtcttgtg atgtactggc tgatttctac gaccagttcg
     5101 ctgaccagtt gcacgagtct caattggaca aaatgccagc acttccggct aaaggtaact
     5161 tgaacctccg tgacatctta gagtcggact tcgcgttcgc gtaatgttta aacgaattcg
     5221 ggctcggtac ccggggatcc tctagagtcg acctgcaggc atgcaagcca aaatcaacgg
     5281 gactttccaa aatgtcgtaa taaccccgcc ccgttgacgc aaatgggcgg taggcgtgta
     5341 cggtgggagg tctatataag cagagctggt ttagtgaacc gtcagatcac tagaagctta
     5401 cgctaccacc atgaacacga ttaacatcgc taagaacgac ttctctgaca tcgaactggc
     5461 tgctatcccg ttaaacactc tggctgacca ttacggtgag cgttcagctc gcggacagtt
     5521 ggcccttgag catgagtctt acgagatggg tgaagcacgc ttccgcaaga tgtttgagtg
     5581 tcaacttaaa gctggtaagg ttgcggataa tgctgccgcc aagcctctca tcactaccct
     5641 actccctaag atgattgccc gcatcaacga ctggtttgag gaagtgaaag ctaagcgcgg
     5701 caggcgcccg acagccttca agttcctaaa agaaatcaag ccggaagccg tagcgtacat
     5761 caccattaag acctctctgg cttgcctaac cagtgctgac aatacaaccg ttcaggctgt
     5821 agcaagcgca atcggtcgga ccattgagga cgaggctcgc ttcggtcgta tccgtgacct
     5881 tgaagctaag cacttcaaga aaaacgttga ggaacaactc aacaagcgcg tagggcacgt
     5941 ctacaagggt ggctctggct ctggctcgag cgtccccctg tatgggttca ccagcatttg
     6001 tgggcggaga ccagagatgg aggcagccgt cagcactatt ccccggttcc tgcagtcaag
     6061 ctccggcagc atgctggacg ggaggttcga tccacagtcc gccgctcact tctttggggt
     6121 ctacgatgga catggcgggt cccaggtggc caactattgc agggagcgca tgcacctggc
     6181 tctggcagag gaaatcgcca aggagaaacc tatgctgtgc gacggagata catggctgga
     6241 aaagtggaag aaagccctgt tcaactcttt tctgcgggtg gactccgaga tcgaatctgt
     6301 cgctcctgag accgtgggct ctacaagtgt ggtcgcagtg gtcttcccat ctcacatttt
     6361 tgtcgcaaat tgcggcgaca gccgggccgt gctgtgcagg ggaaagaccg ccctgccact
     6421 gagcgtggac cataaacccg acagggagga tgaagcagcc cgcatcgagg ctgcaggagg
     6481 caaagtgatc cagtggaacg gagcacgggt gtttggcgtc ctggccatgt cacgcagcat
     6541 tggggatcga tacctgaaac catcaatcat tcccgaccct gaagtgactg ccgtcaagag
     6601 agtgaaagaa gacgattgcc tgatcctggc tagcgacggc gtctgggatg tgatgaccga
     6661 cgaggaagca tgtgagatgg cccgaaagcg gattctgctg tggcataaga aaaatgccgt
     6721 ggctggcgat gctagtctgc tggcagacga gcggagaaag gaagggaaag atcccgccgc
     6781 tatgagtgca gccgaatatc tgtcaaaact ggctattcag aggggcagta aggacaacat
     6841 ctccgtcgtc gtcgtggacc tgaagtgaaa gcccgaaagg aagctgagtt ggctgctgcc
     6901 accgctgagc aataactagc ataacccctt ggggcggccg cttcgagcag acatgataag
     6961 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     7021 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
     7081 caacaacaat tgcattcatt ttatgtttca ggttcagggg gagatgtggg aggttttttt
     7141 aagcaagtaa aacctctaca aatgtggtaa aatcgaattc taatggatcc tctttgcgct
     7201 tgcgttttcc cttgtccaga tagcccagta gctgacattc atccggggtc agcaccgttt
     7261 ctgcggactg gctttctacg tgttccgctt cctttagcag cccttgcgcc ctgagtgctt
     7321 gcggcagcgt gagcttcaaa agaattgcca gctggggcgc cctctggtaa ggttgggaag
     7381 ccctgcaaag taaactggat ggctttcttg ccgccaagga tctgatggcg caggggatca
     7441 agatctgatc aagagacagg atgacggtcg tttcgcatgc ttgaacaaga tggattgcac
     7501 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     7561 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     7621 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     7681 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     7741 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     7801 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     7861 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg cactcggatg
     7921 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     7981 gaactgttcg ccaggctcaa ggcgcgtatg ccggatggtg aggatctcgt cgtgactcat
     8041 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     8101 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     8161 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     8221 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     8281 tggggttcga aatgaccgac caagcgacgc ccaaccggta tcagctcact caaaggcggt
     8341 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     8401 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     8461 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     8521 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     8581 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     8641 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tc
//
