LOCUS       Exported                5786 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  AmtR-A1 gate.
ACCESSION   .
VERSION     .
KEYWORDS    pAmtR-A1
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5786)
  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 5786)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5786
                     /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        2837..2886
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             2936..3655
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2981..3002)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3242..3261)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3589..3610
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3803..4885
                     /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(3822..3841)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4904..5527
                     /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(4977..4996)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(5710..5729)
                     /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 tagggtgtct caaggtgcgt accttgactg atgagtccga aaggacgaaa
     1981 cacccctcta caaataattt tgtttaaaat gttccctaat aatcagcaaa gaggttacta
     2041 gatggcaggc gcagttggtc gtccgcgtcg tagtgcaccg cgtcgtgcag gtaaaaatcc
     2101 gcgtgaagaa attctggatg caagcgcaga actgtttacc cgtcagggtt ttgcaaccac
     2161 cagtacccat cagattgcag atgcagttgg tattcgtcag gcaagcctgt attatcattt
     2221 tccgagcaaa accgaaatct ttctgaccct gctgaaaagc accgttgaac cgagcaccgt
     2281 tctggcagaa gatctgagca ccctggatgc aggtccggaa atgcgtctgt gggcaattgt
     2341 tgcaagcgaa gttcgtctgc tgctgagcac caaatggaat gttggtcgtc tgtatcagct
     2401 gccgattgtt ggtagcgaag aatttgcaga atatcatagc cagcgtgaag cactgaccaa
     2461 tgtttttcgt gatctggcaa ccgaaattgt tggtgatgat ccgcgtgcag aactgccgtt
     2521 tcatattacc atgagcgtta ttgaaatgcg tcgcaatgat ggtaaaattc cgagtccgct
     2581 gagcgcagat agcctgccgg aaaccgcaat tatgctggca gatgcaagcc tggcagttct
     2641 gggtgcaccg ctgcctgcag atcgtgttga aaaaaccctg gaactgatta aacaggcaga
     2701 tgcaaaataa ctcggtacca aagacgaaca ataagacgct gaaaagcgtc ttttttcgtt
     2761 ttggtcctac tcttgtccaa ccaaatgatt cgttaccaat tgacagtttc tatcgatcta
     2821 tagataatgc tagcagctgt caccggatgt gctttccggt ctgatgagtc cgtgaggacg
     2881 aaacagcctc tacaaataat tttgtttaat actagagaaa gaggggaaat actagatggt
     2941 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     3001 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     3061 gctgaccctg aagttcatct gcaccacagg caagctgccc gtgccctggc ccaccctcgt
     3121 gaccaccttc ggctacggcc tgcaatgctt cgcccgctac cccgaccaca tgaagctgca
     3181 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     3241 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     3301 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     3361 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     3421 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     3481 ctaccagcag aacaccccaa tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     3541 tagctaccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     3601 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtaactcgg
     3661 taccaaattc cagaaaagag gcctcccgaa aggggggcct tttttcgttt tggtccaatg
     3721 gcggcgcgcc atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga
     3781 gagtcaattc agggtggtga atatgaaacc agtaacgtta tacgatgtcg cagagtatgc
     3841 cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa
     3901 aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc
     3961 acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct
     4021 gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag
     4081 cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa
     4141 tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc
     4201 cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca
     4261 gacacccatc aacagtatta ttttctccca tgaggacggt acgcgactgg gcgtggagca
     4321 tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc
     4381 ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat
     4441 agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct
     4501 gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc
     4561 aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata
     4621 cgacgatacc gaagatagct catgttatat cccgccgtta accaccatca aacaggattt
     4681 tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt
     4741 gaagggcaat cagctgttgc cagtctcact ggtgaaaaga aaaaccaccc tggcgcccaa
     4801 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     4861 ttcccgactg gaaagcgggc agtgataatc caggaggaaa aaaatgtcca gattagataa
     4921 aagtaaagtg attaacagcg cattagagct gcttaatgag gtcggaatcg aaggtttaac
     4981 aacccgtaaa ctcgcccaga agctaggtgt agagcagcct acattgtatt ggcatgtaaa
     5041 aaataagcgg gctttgctcg acgccttagc cattgagatg ttagataggc accatactca
     5101 cttttgccct ttagaagggg aaagctggca agatttttta cgtaataacg ctaaaagttt
     5161 tagatgtgct ttactaagtc atcgcgatgg agcaaaagta catttaggta cacggcctac
     5221 agaaaaacag tatgaaactc tcgaaaatca attagccttt ttatgccaac aaggtttttc
     5281 actagagaat gcattatatg cactcagcgc tgtggggcat tttactttag gttgcgtatt
     5341 ggaagatcaa gagcatcaag tcgctaaaga agaaagggaa acacctacta ctgatagtat
     5401 gccgccatta ttacgacaag ctatcgaatt atttgatcac caaggtgcag agccagcctt
     5461 cttattcggc cttgaattga tcatatgcgg attagaaaaa caacttaaat gtgaaagtgg
     5521 gtcctaataa ttggtaacga atcagacaat tgacggctcg agggagtagc atagggtttg
     5581 cagaatccct gcttcgtcca tttgacaggc acattatgca tcgatgataa gctgtcaaac
     5641 atgagcagat cctctacgcc ggacgcatcg tggccggcat caccggcgcc acaggtgcgg
     5701 ttgctggcgc ctatatcgcc gacatcaccg atggggaaga tcgggctcgc cacttcgggc
     5761 tcatgagcaa atattttatc tgaggt
//
