LOCUS       Exported                5718 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  LmrA-N1 gate.
ACCESSION   .
VERSION     .
KEYWORDS    pLmrA-N1
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5718)
  AUTHORS   Nielsen AA, Der BS, Shin J, Vaidyanathan P, Paralanov V, Strychalski
            EA, Ross D, Densmore D, Voigt CA
  TITLE     Genetic circuit design automation.
  JOURNAL   Science. 2016 Apr 1;352(6281):aac7341. doi: 10.1126/science.aac7341.
  PUBMED    27034378
REFERENCE   2  (bases 1 to 5718)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5718
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      complement(308..852)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(388..407)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(941..1756)
                     /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     1664..1683
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1748..1767
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        1878..1906
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1912..1931
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     misc_RNA        2769..2818
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             2868..3587
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2913..2934)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3174..3193)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3521..3542
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3735..4817
                     /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     complement(3754..3773)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4836..5459
                     /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     complement(4909..4928)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(5642..5661)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gcttcctcgc tcactgactc gctgcacgag gcagacctca gcgctagcgg agtgtatact
       61 ggcttactat gttggcactg atgagggtgt cagtgaagtg cttcatgtgg caggagaaaa
      121 aaggctgcac cggtgcgtca gcagaatatg tgatacagga tatattccgc ttcctcgctc
      181 actgactcgc tacgctcggt cgttcgactg cggcgagcgg aaatggctta cgaacggggc
      241 ggagatttcc tggaagatgc caggaagata cttaacaggg aagtgagagg gccgcggcaa
      301 agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga cgctcaaatc
      361 agtggtggcg aaacccgaca ggactataaa gataccaggc gtttcccctg gcggctccct
      421 cgtgcgctct cctgttcctg cctttcggtt taccggtgtc attccgctgt tatggccgcg
      481 tttgtctcat tccacgcctg acactcagtt ccgggtaggc agttcgctcc aagctggact
      541 gtatgcacga accccccgtt cagtccgacc gctgcgcctt atccggtaac tatcgtcttg
      601 agtccaaccc ggaaagacat gcaaaagcac cactggcagc agccactggt aattgattta
      661 gaggagttag tcttgaagtc atgcgccggt taaggctaaa ctgaaaggac aagttttggt
      721 gactgcgctc ctccaagcca gttacctcgg ttcaaagagt tggtagctca gagaaccttc
      781 gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc aagagattac gcgcagacca
      841 aaacgatctc aagaagatca tcttattaag gggtctgacg ctcagtggaa cgaaaaatca
      901 atctaaagta tatatgagta aacttggtct gacagttacc ttagaaaaac tcatcgagca
      961 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
     1021 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
     1081 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
     1141 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
     1201 aaagcttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
     1261 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata
     1321 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
     1381 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
     1441 ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
     1501 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
     1561 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
     1621 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     1681 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctcgagcaa gacgtttccc
     1741 gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg
     1801 ttcatgatga tatattttta tcttgtgcaa tgtacatcag agattttgag acacaaggag
     1861 aacgatcgtt ggctgtgttg acaattaatc atcggctcgt ataatgtgtg gaattgtgag
     1921 cgctcacaat taggagtcaa ttaatgtgct tttaattctg atgagacggt gacgtcgaaa
     1981 ctccctctac aaataatttt gtttaatacg ctatggacta tgttttctgc tatggactat
     2041 gttttcacac acgagatgcc tcgatgagct atggtgatag ccgtgaaaaa attctgagcg
     2101 cagcaacccg tctgtttcag ctgcagggtt attatggcac cggtctgaat cagattatca
     2161 aagaaagcgg tgcaccgaaa ggtagcctgt attatcattt tccgggtggt aaagaacagc
     2221 tggcaattga agcagtgaac gaaatgaaag aatatatccg ccagaaaatc gccgattgta
     2281 tggaagcatg taccgatccg gcagaaggta ttcaggcatt tctgaaagaa ctgagctgtc
     2341 agtttagctg taccgaagat attgaaggtc tgccggttgg tctgctggca gcagaaacca
     2401 gcctgaaaag cgaaccgctg cgtgaagcat gtcatgaagc atataaagaa tgggccagcg
     2461 tgtatgaaga aaaactgcgt cagaccggtt gtagcgaaag ccgtgcaaaa gaagcaagca
     2521 ccgttgttaa tgcaatgatt gaaggtggta ttctgctgag cctgaccgca aaaaatagca
     2581 caccgctgct gcatattagc agctgtattc cggatctgct gaaacgttaa taaggttgaa
     2641 aaataaaaac ggcgctaaaa agcgccgttt ttttgacggt ggtatactcg ctcattcact
     2701 aggtctgatt cgttaccaat tgacagctgg tggtcgaatc aagataatag accagtcact
     2761 atatttagct gtcaccggat gtgctttccg gtctgatgag tccgtgagga cgaaacagcc
     2821 tctacaaata attttgttta atactagaga aagaggggaa atactagatg gtgagcaagg
     2881 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     2941 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     3001 tgaagttcat ctgcaccaca ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct
     3061 tcggctacgg cctgcaatgc ttcgcccgct accccgacca catgaagctg cacgacttct
     3121 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     3181 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     3241 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     3301 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     3361 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     3421 agaacacccc aatcggcgac ggccccgtgc tgctgcccga caaccactac cttagctacc
     3481 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     3541 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaactc ggtaccaaat
     3601 tccagaaaag aggcctcccg aaaggggggc cttttttcgt tttggtccaa tggcggcgcg
     3661 ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat
     3721 tcagggtggt gaatatgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct
     3781 cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg
     3841 aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac
     3901 tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc
     3961 cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg
     4021 tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg
     4081 cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg
     4141 tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca
     4201 tcaacagtat tattttctcc catgaggacg gtacgcgact gggcgtggag catctggtcg
     4261 cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc
     4321 tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac
     4381 gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg
     4441 gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg
     4501 ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata
     4561 ccgaagatag ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc
     4621 tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca
     4681 atcagctgtt gccagtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa
     4741 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     4801 tggaaagcgg gcagtgataa tccaggagga aaaaaatgtc cagattagat aaaagtaaag
     4861 tgattaacag cgcattagag ctgcttaatg aggtcggaat cgaaggttta acaacccgta
     4921 aactcgccca gaagctaggt gtagagcagc ctacattgta ttggcatgta aaaaataagc
     4981 gggctttgct cgacgcctta gccattgaga tgttagatag gcaccatact cacttttgcc
     5041 ctttagaagg ggaaagctgg caagattttt tacgtaataa cgctaaaagt tttagatgtg
     5101 ctttactaag tcatcgcgat ggagcaaaag tacatttagg tacacggcct acagaaaaac
     5161 agtatgaaac tctcgaaaat caattagcct ttttatgcca acaaggtttt tcactagaga
     5221 atgcattata tgcactcagc gctgtggggc attttacttt aggttgcgta ttggaagatc
     5281 aagagcatca agtcgctaaa gaagaaaggg aaacacctac tactgatagt atgccgccat
     5341 tattacgaca agctatcgaa ttatttgatc accaaggtgc agagccagcc ttcttattcg
     5401 gccttgaatt gatcatatgc ggattagaaa aacaacttaa atgtgaaagt gggtcctaat
     5461 aattggtaac gaatcagaca attgacggct cgagggagta gcatagggtt tgcagaatcc
     5521 ctgcttcgtc catttgacag gcacattatg catcgatgat aagctgtcaa acatgagcag
     5581 atcctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc ggttgctggc
     5641 gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc
     5701 aaatatttta tctgaggt
//
