LOCUS       sequence-273529-        6206 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Reporter plasmid - Expresses three fluorescent reporters from three 
            different ECF promoters.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6206)
  AUTHORS   Pinto F, Thornton EL, Wang B
  TITLE     An expanded library of orthogonal split inteins enables modular 
            multi-peptide assemblies.
  JOURNAL   Nat Commun. 2020 Mar 23;11(1):1529. doi: 10.1038/s41467-020-15272-2.
  PUBMED    32251274
REFERENCE   2  (bases 1 to 6206)
  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: "Nat 
            Commun."; date: "2020-03-23"; volume: "11(1):1529. doi"; pages: " 
            10.1038/s41467-020-15272-2"
COMMENT     Sequence Label: pFP.E227
FEATURES             Location/Qualifiers
     source          1..6206
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     51..69
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(73..116)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    119..140
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     CDS             complement(147..164)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(165..872)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(440..459)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     709..727
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     RBS             879..890
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      978..1049
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1065..1092
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     RBS             1178..1189
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1895..1912
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1935..2006
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2022..2049
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     RBS             2135..2146
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2153..2866
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(2183..2211)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2806..2826
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2889..2960
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2976..3003
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    3010..3030
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      3031..3088
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     primer_bind     complement(3112..3129)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(3391..4341)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      4389..4611
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(5103..5197)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(5227..6018,6019..6087))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5850..5869
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6088..6192)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta
       61 tcacgaggca gaatttcaga taaaaaaaat ccttagcttt cgctaaggat gatttctgga
      121 attcgcggcc gcttctagag ttattagtgg tgatggtgat gatgcttgta cagctcgtcc
      181 atgccgccgg tggagtggcg gccctcggcg cgttcgtact gttccacgat ggtgtagtcc
      241 tcgttgtggg aggtgatgtc caacttgatg ttgacgttgt aggcgccggg cagctgcacg
      301 ggcttcttgg ccttgtaggt ggtcttgacc tcagcgtcgt agtggccgcc gtccttcagc
      361 ttcagcctct gcttgatctc gcccttcagg gcgccgtcct cggggtacat ccgctcggag
      421 gaggcctccc agcccatggt cttcttctgc attacggggc cgtcggaggg gaagttggtg
      481 ccgcgcagct tcaccttgta gatgaactcg ccgtcctgca aggaggagtc ctgggtcacg
      541 gtcaccacgc cgccgtcctc gaagttcatc acgcgctccc acttgaagcc ctcggggaag
      601 gacagcttca agtagtcggg gatgtcggcg gggtgcttca cgtaggcctt ggagccgtac
      661 atgaactgag gggacaggat gtcccaggcg aagggcaggg ggccaccctt ggtcaccttc
      721 agcttggcgg tctgggtgcc ctcgtagggg cggccctcgc cctcgccctc gatctcgaac
      781 tcgtggccgt tcacggagcc ctccatgtgc accttgaagc gcatgaactc cttgatgatg
      841 gccatgttgt cttcttcgcc cttgctcacc atctagtatt tctcctcttt aatctctagt
      901 aagttagttc gttgcatcgg ccgccagggt tacaccgtcg gtgggtttct tttcatccaa
      961 gtactagagc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt
     1021 ttatctgttg tttgtcggtg aacgctctct actagagtca cactggctca ccttcgggtg
     1081 ggcctttctg cgtttatata ctagagcagt acaaaatttt ttagatgcgt ttggtgaacc
     1141 aaactcttac tcgactcgtg tcagtatact agagattaaa gaggagaaat actagatgag
     1201 cgaactgatc aaagagaaca tgcacatgaa gctgtacatg gaaggcaccg ttgacaacca
     1261 ccactttaag tgcacgtctg agggtgaggg taagccgtac gaaggcaccc aaaccatgcg
     1321 tatcaaagtt gtggagggcg gtccactgcc gttcgctttt gacattctgg cgaccagctt
     1381 cctgtacggt tccaaaacgt tcattaacca tactcagggc attccggatt tcttcaaaca
     1441 gagctttccg gaaggtttca cctgggagcg tgtcaccacg tatgaagatg gtggtgtgtt
     1501 gaccgccacc caagatacct ccctgcaaga tggctgtctg atctataacg tgaaaattcg
     1561 tggcgtcaac tttacgagca atggtccggt gatgcagaag aaaaccctgg gttgggaggc
     1621 gtttacggaa accctgtatc cggccgatgg tggcctggag ggccgtaacg acatggcact
     1681 gaagctggtt ggtggcagcc atttgatcgc aaatatcaag acgacgtacc gcagcaagaa
     1741 accggcgaaa aatctgaaga tgccgggtgt ttactatgtc gactaccgtc tggaacgcat
     1801 taaagaagcg aataatgaga cttacgtgga gcagcacgag gttgcagtcg cgcgctattg
     1861 cgacttgcct agcaagctgg gtcataaact gaatcatcat caccatcacc actaataata
     1921 ctagagccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta
     1981 tctgttgttt gtcggtgaac gctctctact agagtcacac tggctcacct tcgggtgggc
     2041 ctttctgcgt ttatatacta gaggcgcgga taaaaatttc atttgcccgc gacggattcc
     2101 ccgcccatct atcgttgaac ccatactaga gattaaagag gagaaatact agatgcgtaa
     2161 aggagaagaa cttttcactg gagttgtccc aattcttgtt gaattagatg gtgatgttaa
     2221 tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat gcaacatacg gaaaacttac
     2281 ccttaaattt atttgcacta ctggaaaact acctgttcca tggccaacac ttgtcactac
     2341 tttcggttat ggtgttcaat gctttgcgag atacccagat catatgaaac agcatgactt
     2401 tttcaagagt gccatgcccg aaggttatgt acaggaaaga actatatttt tcaaagatga
     2461 cgggaactac aagacacgtg ctgaagtcaa gtttgaaggt gatacccttg ttaatagaat
     2521 cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt cttggacaca aattggaata
     2581 caactataac tcacacaatg tatacatcat ggcagacaaa caaaagaatg gaatcaaagt
     2641 taacttcaaa attagacaca acattgaaga tggaagcgtt caactagcag accattatca
     2701 acaaaatact ccaattggcg atggccctgt ccttttacca gacaaccatt acctgtccac
     2761 acaatctgcc ctttcgaaag atcccaacga aaagagagac cacatggtcc ttcttgagtt
     2821 tgtaacagct gctgggatta cacatggcat ggatgaacta tacaaataat aatactagag
     2881 ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     2941 gtttgtcggt gaacgctctc tactagagtc acactggctc accttcgggt gggcctttct
     3001 gcgtttatat actagtagcg gccgctgcag tccggcaaaa aagggcaagg tgtcaccacc
     3061 ctgccctttt tctttaaaac cgaaaagatt acttcgcgtt atgcaggctt cctcgctcac
     3121 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     3181 aatctcgagg ttacattgtc gatctgttca tggtgaacag ctttgaatgc accaaaaact
     3241 cgtaaaagct ctgatgtatc tatctttttt acaccgtttt catctgtgca tatggacagt
     3301 tttccctttg atatgtaacg gtgaacagtt gttctacttt tgtttgttag tcttgatgct
     3361 tcactgatag atacaagagc cataagaacc tcagatcctt ccgtatttag ccagtatgtt
     3421 ctctagtgtg gttcgttgtt tttgcgtgag ccatgagaac gaaccattga gatcatactt
     3481 actttgcatg tcactcaaaa attttgcctc aaaactggtg agctgaattt ttgcagttaa
     3541 agcatcgtgt agtgtttttc ttagtccgtt atgtaggtag gaatctgatg taatggttgt
     3601 tggtattttg tcaccattca tttttatctg gttgttctca agttcggtta cgagatccat
     3661 ttgtctatct agttcaactt ggaaaatcaa cgtatcagtc gggcggcctc gcttatcaac
     3721 caccaatttc atattgctgt aagtgtttaa atctttactt attggtttca aaacccattg
     3781 gttaagcctt ttaaactcat ggtagttatt ttcaagcatt aacatgaact taaattcatc
     3841 aaggctaatc tctatatttg ccttgtgagt tttcttttgt gttagttctt ttaataacca
     3901 ctcataaatc ctcatagagt atttgttttc aaaagactta acatgttcca gattatattt
     3961 tatgaatttt tttaactgga aaagataagg caatatctct tcactaaaaa ctaattctaa
     4021 tttttcgctt gagaacttgg catagtttgt ccactggaaa atctcaaagc ctttaaccaa
     4081 aggattcctg atttccacag ttctcgtcat cagctctctg gttgctttag ctaatacacc
     4141 ataagcattt tccctactga tgttcatcat ctgagcgtat tggttataag tgaacgatac
     4201 cgtccgttct ttccttgtag ggttttcaat cgtggggttg agtagtgcca cacagcataa
     4261 aattagcttg gtttcatgct ccgttaagtc atagcgacta atcgctagtt catttgcttt
     4321 gaaaacaact aattcagaca tacatctcaa ttggtctagg tgattttaat cactatacca
     4381 attgagatgg gctagtcaat gataattaca tgtccttttc ctttgagttg tgggtatctg
     4441 taaattctgc tagacctttg ctggaaaact tgtaaattct gctagaccct ctgtaaattc
     4501 cgctagacct ttgtgtgttt tttttgttta tattcaagtg gttataattt atagaataaa
     4561 gaaagaataa aaaaagataa aaagaataga tcccagccct gtgtataact cactacttta
     4621 gtcagttccg cagtattaca aaaggatgtc gcaaacgctg tttgctcctc tacaaaacag
     4681 accttaaaac cctaaaggct taagtagcac cctcgcaagc tcgggcaaat cgctgaatat
     4741 tccttttgtc tccgaccatc aggcacctga gtcgctgtct ttttcgtgac attcagttcg
     4801 ctgcgctcac ggctctggca gtgaatgggg gtaaatggca ctacaggcgc cttttatgga
     4861 ttcatgcaag gaaactaccc ataatacaag aaaagcccgt cacgggcttc tcagggcgtt
     4921 ttatggcggg tctgctatgt ggtgctatct gactttttgc tgttcagcag ttcctgccct
     4981 ctgattttcc agtctgacca cttcggatta tcccgtgaca ggtcattcag actggctaat
     5041 gcacccagta aggcagcggt atcatcaaca ggcttacccg tcttactgtc cctagtgctt
     5101 ggattctcac caataaaaaa cgcccggcgg caaccgagcg ttctgaacaa atccagatgg
     5161 agttctgagg tcattactgg atctatcaac aggagtccaa gcgagctcgt aaacttggtc
     5221 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     5281 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     5341 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     5401 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     5461 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     5521 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     5581 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     5641 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     5701 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     5761 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     5821 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     5881 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     5941 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     6001 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     6061 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     6121 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     6181 aggggttccg cgcacatttc ccatgg
//
