LOCUS       sequence-362687-        4920 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RPU standard plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4920)
  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 4920)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science."; 
            date: "2016-04-1"; volume: "352(6281):aac7341. doi"; pages: " 
            10.1126/science.aac7341"
COMMENT     Sequence Label: pAN1717
FEATURES             Location/Qualifiers
     source          1..4920
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        21..55
                     /label=BBa_J23101
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.7"
     misc_RNA        62..111
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             join(161..163,164..166,167..880)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(206..227)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(467..486)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     814..835
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1028..2110
                     /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(1047..1066)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2129..2752
                     /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(2202..2221)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(2935..2954)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     rep_origin      complement(3319..3864)
                     /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(3399..3418)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3953..4768)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3953..4768)
                     /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     4676..4695
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4760..4779
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 cgcttgataa gtccctaact tttacagcta gctcagtcct aggtattatg ctagcctgaa
       61 gctgtcaccg gatgtgcttt ccggtctgat gagtccgtga ggacgaaaca gcctctacaa
      121 ataattttgt ttaatactag agaaagaggg gaaatactag atggtgagca agggcgagga
      181 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
      241 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
      301 catctgcacc acaggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccttcggcta
      361 cggcctgcaa tgcttcgccc gctaccccga ccacatgaag ctgcacgact tcttcaagtc
      421 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
      481 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
      541 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
      601 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
      661 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
      721 cccaatcggc gacggccccg tgctgctgcc cgacaaccac taccttagct accagtccgc
      781 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
      841 cgccgggatc actctcggca tggacgagct gtacaagtaa ctcggtacca aattccagaa
      901 aagaggcctc ccgaaagggg ggcctttttt cgttttggtc caatggcggc gcgccatcga
      961 atggcgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt
     1021 ggtgaatatg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca
     1081 gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt
     1141 ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg
     1201 caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca
     1261 aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat
     1321 ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg
     1381 cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc
     1441 tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag
     1501 tattattttc tcccatgagg acggtacgcg actgggcgtg gagcatctgg tcgcattggg
     1561 tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct
     1621 ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg
     1681 cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt
     1741 tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac
     1801 cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga
     1861 tagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca
     1921 aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct
     1981 gttgccagtc tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc
     2041 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     2101 cgggcagtga taatccagga ggaaaaaaat gtccagatta gataaaagta aagtgattaa
     2161 cagcgcatta gagctgctta atgaggtcgg aatcgaaggt ttaacaaccc gtaaactcgc
     2221 ccagaagcta ggtgtagagc agcctacatt gtattggcat gtaaaaaata agcgggcttt
     2281 gctcgacgcc ttagccattg agatgttaga taggcaccat actcactttt gccctttaga
     2341 aggggaaagc tggcaagatt ttttacgtaa taacgctaaa agttttagat gtgctttact
     2401 aagtcatcgc gatggagcaa aagtacattt aggtacacgg cctacagaaa aacagtatga
     2461 aactctcgaa aatcaattag cctttttatg ccaacaaggt ttttcactag agaatgcatt
     2521 atatgcactc agcgctgtgg ggcattttac tttaggttgc gtattggaag atcaagagca
     2581 tcaagtcgct aaagaagaaa gggaaacacc tactactgat agtatgccgc cattattacg
     2641 acaagctatc gaattatttg atcaccaagg tgcagagcca gccttcttat tcggccttga
     2701 attgatcata tgcggattag aaaaacaact taaatgtgaa agtgggtcct aataattggt
     2761 aacgaatcag acaattgacg gctcgaggga gtagcatagg gtttgcagaa tccctgcttc
     2821 gtccatttga caggcacatt atgcatcgat gataagctgt caaacatgag cagatcctct
     2881 acgccggacg catcgtggcc ggcatcaccg gcgccacagg tgcggttgct ggcgcctata
     2941 tcgccgacat caccgatggg gaagatcggg ctcgccactt cgggctcatg agcaaatatt
     3001 ttatctgagg tgcttcctcg ctcactgact cgctgcacga ggcagacctc agcgctagcg
     3061 gagtgtatac tggcttacta tgttggcact gatgagggtg tcagtgaagt gcttcatgtg
     3121 gcaggagaaa aaaggctgca ccggtgcgtc agcagaatat gtgatacagg atatattccg
     3181 cttcctcgct cactgactcg ctacgctcgg tcgttcgact gcggcgagcg gaaatggctt
     3241 acgaacgggg cggagatttc ctggaagatg ccaggaagat acttaacagg gaagtgagag
     3301 ggccgcggca aagccgtttt tccataggct ccgcccccct gacaagcatc acgaaatctg
     3361 acgctcaaat cagtggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     3421 tggcggctcc ctcgtgcgct ctcctgttcc tgcctttcgg tttaccggtg tcattccgct
     3481 gttatggccg cgtttgtctc attccacgcc tgacactcag ttccgggtag gcagttcgct
     3541 ccaagctgga ctgtatgcac gaaccccccg ttcagtccga ccgctgcgcc ttatccggta
     3601 actatcgtct tgagtccaac ccggaaagac atgcaaaagc accactggca gcagccactg
     3661 gtaattgatt tagaggagtt agtcttgaag tcatgcgccg gttaaggcta aactgaaagg
     3721 acaagttttg gtgactgcgc tcctccaagc cagttacctc ggttcaaaga gttggtagct
     3781 cagagaacct tcgaaaaacc gccctgcaag gcggtttttt cgttttcaga gcaagagatt
     3841 acgcgcagac caaaacgatc tcaagaagat catcttatta aggggtctga cgctcagtgg
     3901 aacgaaaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccttagaaaa
     3961 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     4021 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     4081 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     4141 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     4201 gtgagaatgg caaaagctta tgcatttctt tccagacttg ttcaacaggc cagccattac
     4261 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     4321 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     4381 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     4441 atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag
     4501 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     4561 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     4621 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
     4681 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctggagc
     4741 aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag
     4801 acagttttat tgttcatgat gatatatttt tatcttgtgc aatgtacatc agagattttg
     4861 agacacaacc aattattgaa ggcctcccta acggggggcc tttttttgtt tctggtctcc
//
