LOCUS       sequence-148248-        5778 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  inducible RFP expression vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5778)
  AUTHORS   Abudayyeh OO, Gootenberg JS, Konermann S, Joung J, Slaymaker IM, Cox
            DB, Shmakov S, Makarova KS, Semenova E, Minakhin L, Severinov K, 
            Regev A, Lander ES, Koonin EV, Zhang F
  TITLE     C2c2 is a single-component programmable RNA-guided RNA-targeting 
            CRISPR effector.
  JOURNAL   Science. 2016 Jun 2. pii: aaf5573.
  PUBMED    27256883
REFERENCE   2  (bases 1 to 5778)
  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. 
            2016 Jun 2. pii: aaf5573."
COMMENT     Sequence Label: pC008 - pBR322 with tetR-inducible RFP
FEATURES             Location/Qualifiers
     source          1..5778
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(81..1271)
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     900..919
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        complement(join(1319..1324,1325..1341,1342..1347))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1374..1392)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1460..1564
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1565..1633,1634..2425)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1783..1802)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2596..3184
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3085..3104
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3338..3355
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(3430..4056)
                     /codon_start=1
                     /gene="tetR"
                     /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="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     3961..3980
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        4072..4127
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    4108..4126
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             4145..4156
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             4163..4840
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(4853..4872)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5013..5035
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5029..5220)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 attcacagtt ctccgcaaga attgattggc tccaattctt ggagtggtga atccgttagc
       61 gaggtgccgc cggcttccat tcaggtcgag gtggcccggc tccatgcacc gcgacgcaac
      121 gcggggaggc agacaaggta tagggcggcg cctacaatcc atgccaaccc gttccatgtg
      181 ctcgccgagg cggcataaat cgccgtgacg atcagcggtc caatgatcga agttaggctg
      241 gtaagagccg cgagcgatcc ttgaagctgt ccctgatggt cgtcatctac ctgcctggac
      301 agcatggcct gcaacgcggg catcccgatg ccgccggaag cgagaagaat cataatgggg
      361 aaggccatcc agcctcgcgt cgcgaacgcc agcaagacgt agcccagcgc gtcggccgcc
      421 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
      481 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
      541 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
      601 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
      661 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgacgctc tcccttatgc
      721 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
      781 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
      841 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
      901 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
      961 atgcgtccgg cgtagaggat ccacaggacg ggtgtggtcg ccatgatcgc gtagtcgata
     1021 gtggctccaa gtagcgaagc gagcaggact gggcggcggc caaagcggtc ggacagtgct
     1081 ccgagaacgg gtgcgcatag aaattgcatc aacgcatata gcgctagcag cacgccatag
     1141 tgactggcga tgctgtcgga atggacgata tcccgcaaga ggcccggcag taccggcata
     1201 accaagccta tgcctacagc atccagggtg acggtgccga ggatgacgat gagcgcattg
     1261 ttagatttca tacacggtgc ctgactgcgt tagcaattta actgtgataa actaccgcat
     1321 taaagcttat cgatgataag ctgtcaaaca tgagaattct tgaagacgaa agggcctcgt
     1381 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     1441 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     1501 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     1561 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     1621 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     1681 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     1741 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     1801 atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     1861 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     1921 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     1981 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     2041 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     2101 gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     2161 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     2221 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     2281 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     2341 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     2401 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     2461 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     2521 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     2581 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     2641 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     2701 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     2761 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     2821 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     2881 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     2941 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     3001 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     3061 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     3121 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     3181 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     3241 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     3301 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     3361 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     3421 atggcatgct taagacccac tttcacattt aagttgtttt tctaatccgc atatgatcaa
     3481 ttcaaggccg aataagaagg ctggctctgc accttggtga tcaaataatt cgatagcttg
     3541 tcgtaataat ggcggcatac tatcagtagt aggtgtttcc ctttcttctt tagcgacttg
     3601 atgctcttga tcttccaata cgcaacctaa agtaaaatgc cccacagcgc tgagtgcata
     3661 taatgcattc tctagtgaaa aaccttgttg gcataaaaag gctaattgat tttcgagagt
     3721 ttcatactgt ttttctgtag gccgtgtacc taaatgtact tttgctccat cgcgatgact
     3781 tagtaaagca catctaaaac ttttagcgtt attacgtaaa aaatcttgcc agctttcccc
     3841 ttctaaaggg caaaagtgag tatggtgcct atctaacatc tcaatggcta aggcgtcgag
     3901 caaagcccgc ttatttttta catgccaata caatgtaggc tgctctacac ctagcttctg
     3961 ggcgagttta cgggttgtta aaccttcgat tccgacctca ttaagcagct ctaatgcgct
     4021 gttaatcact ttacttttat ctaatctaga catcattaat tcctaatttt tgttgacact
     4081 ctatcgttga tagagttatt ttaccactcc ctatcagtga tagagaaaag aattcaaaag
     4141 atctaaagag gagaaaggat ctatggcgag tagcgaagac gttatcaaag agttcatgcg
     4201 tttcaaagtt cgtatggaag gttccgttaa cggtcacgag ttcgaaatcg aaggtgaagg
     4261 tgaaggtcgt ccgtacgaag gtacccagac cgctaaactg aaagttacca aaggtggtcc
     4321 gctgccgttc gcttgggaca tcctgtcccc gcagttccag tacggttcca aagcttacgt
     4381 taaacacccg gctgacatcc cggactacct gaaactgtcc ttcccggaag gtttcaaatg
     4441 ggaacgtgtt atgaacttcg aagacggtgg tgttgttacc gttacccagg actcctccct
     4501 gcaagacggt gagttcatct acaaagttaa actgcgtggt accaacttcc cgtccgacgg
     4561 tccggttatg cagaaaaaaa ccatgggttg ggaagcttcc accgaacgta tgtacccgga
     4621 agacggtgct ctgaaaggtg aaatcaaaat gcgtctgaaa ctgaaagacg gtggtcacta
     4681 cgacgctgaa gttaaaacca cctacatggc taaaaaaccg gttcagctgc cgggtgctta
     4741 caaaaccgac atcaaactgg acatcacctc ccacaacgaa gactacacca tcgttgaaca
     4801 gtacgaacgt gctgaaggtc gtcactccac cggtgcttaa gtgcactctc agtacaatct
     4861 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     4921 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4981 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     5041 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     5101 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     5161 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     5221 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     5281 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     5341 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     5401 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     5461 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     5521 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     5581 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     5641 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtggcc aggacccaac
     5701 gctgcccgag atgcgccgcg tgcggctgct ggagatggcg gacgcgatgg atatgttctg
     5761 ccaagggttg gtttgcgc
//
