LOCUS       sequence-307626-        7576 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Derivative of pSEVA291 carrying the bxb1 gene under the control of a
            rhamnose-inducible promoter and mCherry flanked by attB/P sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7576)
  AUTHORS   Hollerer S, Papaxanthos L, Gumpinger AC, Fischer K, Beisel C, 
            Borgwardt K, Benenson Y, Jeschek M
  TITLE     Large-scale DNA-based phenotypic recording and deep learning enable 
            highly accurate sequence-function mapping.
  JOURNAL   Nat Commun. 2020 Jul 15;11(1):3551. doi: 10.1038/s41467-020-17222-4.
  PUBMED    32669542
REFERENCE   2  (bases 1 to 7576)
  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: "2020-07-15"; volume: "11(1):3551. doi"; pages: " 
            10.1038/s41467-020-17222-4"
COMMENT     Sequence Label: pASPIre1
FEATURES             Location/Qualifiers
     source          1..7576
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(15..863)
                     /codon_start=1
                     /gene="E. coli rhaR"
                     /product="transcriptional activator of rhaR and rhaS"
                     /label=rhaR
                     /note="needed for rhamnose-dependent activation of the rhaB
                     promoter"
                     /translation="MAHQLKLLKDDFFASDQQAVAVADRYPQDVFAEHTHDFCELVIVW
                     RGNGLHVLNDRPYRITRGDLFYIHADDKHSYASVNDLVLQNIIYCPERLKLNLDWQGAI
                     PGFNASAGQPHWRLGSMGMAQARQVIGQLEHESSQHVPFANEMAELLFGQLVMLLNRHR
                     YTSDSLPPTSSETLLDKLITRLAASLKSPFALDKFCDEASCSERVLRQQFRQQTGMTIN
                     QYLRQVRVCHAQYLLQHSRLLISDISTECGFEDSNYFSVVFTRETGMMPSQWRHLNSQK
                     D"
     CDS             complement(937..1773)
                     /codon_start=1
                     /gene="E. coli rhaS"
                     /product="positive regulator of the rhaB promoter
                     "
                     /label=rhaS
                     /note="activated by L-rhamnose"
                     /translation="MTVLHSVDFFPSGNASVAIEPRLPQADFPEHHHDFHEIVIVEHGT
                     GIHVFNGQPYTITGGTVCFVRDHDRHLYEHTDNLCLTNVLYRSPDRFQFLAGLNQLLPQ
                     ELDGQYPSHWRVNHSVLQQVRQLVAQMEQQEGENDLPSTASREILFMQLLLLLRKSSLQ
                     ENLENSASRLNLLLAWLEDHFADEVNWDAVADQFSLSLRTLHRQLKQQTGLTPQRYLNR
                     LRLMKARHLLRHSEASVTDIAYRCGFSDSNHFSTLFRREFNWSPRDIRQGRDGFLQ"
     protein_bind    1928..1977
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attB site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
     RBS             1996..2018
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2026..2733
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MHSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2171..2189)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2439..2458
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    2792..2839
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     promoter        complement(2879..2913)
                     /label=BBa_J23118
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.56"
     promoter        2955..3073
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring 
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
     CDS             4626..4643
                     /codon_start=1
                     /product="tetracysteine peptide that binds biarsenical 
                     labeling reagents"
                     /label=tetracysteine tag
                     /translation="CCPGCC"
     CDS             4644..4676
                     /codon_start=1
                     /product="C-terminal peptide that mediates degradation in 
                     bacteria through the ClpXP and ClpAP proteases (McGinness 
                     et al., 2006)"
                     /label=ssrA tag
                     /note="wild-type LAA variant"
                     /translation="AANDENYALAA"
     primer_bind     complement(4710..4732)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     terminator      4747..4833
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             5013..5204
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5198..5220)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5306..5446
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5361..5380
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5461..5478)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5632..6220)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5712..5731)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     oriT            6256..6364
                     /note="incP origin of transfer"
     CDS             complement(6531..7346)
                     /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"
     primer_bind     7338..7357
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     terminator      complement(7478..7572)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
ORIGIN
        1 actagtaagc ttaattaatc tttctgcgaa ttgagatgac gccactggct gggcatcatc
       61 ccggtttccc gggtaaacac caccgaaaaa tagttactat cttcaaagcc acattcggtc
      121 gaaatatcac tgattaacag gcggctatgc tggagaagat attgcgcatg acacactctg
      181 acctgtcgca gatattgatt gatggtcatt ccagtctgct ggcgaaattg ctgacgcaaa
      241 acgcgctcac tgcacgatgc ctcatcacaa aatttatcca gcgcaaaggg acttttcagg
      301 ctagccgcca gccgggtaat cagcttatcc agcaacgttt cgctggatgt tggcggcaac
      361 gaatcactgg tgtaacgatg gcgattcagc aacatcacca actgcccgaa cagcaactca
      421 gccatttcgt tagcaaacgg cacatgctga ctactttcat gctcaagctg accgataacc
      481 tgccgcgcct gcgccatccc catgctacct aagcgccagt gtggttgccc tgcgctggcg
      541 ttaaatcccg gaatcgcccc ctgccagtca agattcagct tcagacgctc cgggcaataa
      601 ataatattct gcaaaaccag atcgttaacg gaagcgtagg agtgtttatc gtcagcatga
      661 atgtaaaaga gatcgccacg ggtaatgcga taagggcgat cgttgagtac atgcaggcca
      721 ttaccgcgcc agacaatcac cagctcacaa aaatcatgtg tatgttcagc aaagacatct
      781 tgcggataac ggtcagccac agcgactgcc tgctggtcgc tggcaaaaaa atcatctttg
      841 agaagtttta actgatgcgc caccgtggct acctcggcca gagaacgaag ttgattattc
      901 gcaatatggc gtacaaatac gttgagaaga ttcgcgttat tgcagaaagc catcccgtcc
      961 ctggcgaata tcacgcggtg accagttaaa ctctcggcga aaaagcgtcg aaaagtggtt
     1021 actgtcgctg aatccacagc gataggcgat gtcagtaacg ctggcctcgc tgtggcgtag
     1081 cagatgtcgg gctttcatca gtcgcaggcg gttcaggtat cgctgaggcg tcagtcccgt
     1141 ttgctgctta agctgccgat gtagcgtacg cagtgaaaga gaaaattgat ccgccacggc
     1201 atcccaattc acctcatcgg caaaatggtc ctccagccag gccagaagca agttgagacg
     1261 tgatgcgctg ttttccaggt tctcctgcaa actgctttta cgcagcaaga gcagtaattg
     1321 cataaacaag atctcgcgac tggcggtcga gggtaaatca ttttcccctt cctgctgttc
     1381 catctgtgca accagctgtc gcacctgctg caatacgctg tggttaacgc gccagtgaga
     1441 cggatactgc ccatccagct cttgtggcag caactgattc agcccggcga gaaactgaaa
     1501 tcgatccggc gagcgataca gcacattggt cagacacaga ttatcggtat gttcatacag
     1561 atgccgatca tgatcgcgta cgaaacagac cgtgccaccg gtgatggtat agggctgccc
     1621 attaaacaca tgaatacccg tgccatgttc gacaatcaca atttcatgaa aatcatgatg
     1681 atgttcagga aaatccgcct gcgggagccg gggttctatc gccacggacg cgttaccaga
     1741 cggaaaaaaa tccacactat gtaatacggt catactggcc tcctgatgtc gtcaacacgg
     1801 cgaaatagta atcacgaggt caggttctta ccttaaattt tcgacggaaa accacgtaaa
     1861 aaacgtcgat ttttcaagat acagcgtgaa ttttcaggaa atgcggtgag catcacatct
     1921 gcagtaatcg gccggcttgt cgacgacggc ggtctccgtc gtcaggatca tccgggccca
     1981 tgggatatct ataattttgt ttaactttaa gaaggagata tgcacatgca tagcaagggc
     2041 gaagaagata acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc
     2101 tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
     2161 acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc
     2221 ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc
     2281 gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag
     2341 gacggcggcg tggtgaccgt gacccaggac tcctccttgc aggacggcga gttcatctac
     2401 aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagaca
     2461 atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag
     2521 atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc
     2581 tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac
     2641 atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc
     2701 cactccaccg gcggcatgga cgagctgtac aagaggcctt aagcaaaaaa ccccgcttcg
     2761 gcggggtttt ttcgctgcga tatctaataa gggtttgtac cgtacaccac tgagaccgcg
     2821 gtggttgacc agacaaacca cgaaggttct gttaagtaac tgaacccaat gtcgttaggc
     2881 tagcacaata cctaggactg agctagccgt caatgacgct tacctcttaa gaggtcactg
     2941 acctaacagg atcccaccac aattcagcaa attgtgaaca tcatcacgtt catctttccc
     3001 tggttgccaa tggcccattt tcctgtcagt aacgagaagg tcgcgaattc aggcgctttt
     3061 tagactggtc gtaatgaagg gtaccataaa tgcttcaata atattgaaaa ggattctaga
     3121 tgcgtgcact ggttgttatt cgtctgagcc gtgttaccga tgcaaccacc agtccggaac
     3181 gtcagctgga aagctgtcag cagctgtgtg cacagcgtgg ttgggatgtt gttggtgttg
     3241 cagaggatct ggatgttagc ggtgcagttg atccgtttga tcgtaaacgt cgtccgaatc
     3301 tggcacgttg gctggcattt gaagaacagc cgtttgatgt tattgttgcc tatcgtgttg
     3361 atcgtctgac ccgtagcatt cgtcatctgc aacagctggt tcattgggca gaagatcata
     3421 aaaaactggt tgtgagcgca accgaagcac attttgatac caccaccccg tttgcagcag
     3481 ttgttattgc actgatgggc accgttgcac agatggaact ggaagcaatt aaagaacgta
     3541 atcgtagcgc agcccatttt aacattcgtg caggtaaata tcgtggtagc ctgcctccgt
     3601 ggggttatct gccgacgcgt gtagatggtg aatggcgtct ggttcctgac ccggttcagc
     3661 gtgaacgtat tctggaagta tatcatcgtg tggtggataa tcatgaaccg ctgcatctgg
     3721 ttgcacatga tctgaatcgt cgtggtgttc tgagtcccaa agattatttt gctcagctgc
     3781 aaggtcgtga accgcagggt cgtgaatggt ctgcaaccgc actgaaacgt agcatgatta
     3841 gcgaagcaat gctgggttat gcaaccctga atggtaaaac cgttcgtgat gatgatggtg
     3901 caccgctggt tcgtgcagaa ccgattctga cacgtgaaca gctggaagca ctgcgtgccg
     3961 aactggttaa aaccagccgt gcaaaaccgg cagttagcac cccgagcctg ctgctgcgtg
     4021 ttctgttttg tgcagtttgt ggtgaaccgg catacaaatt tgccggtggt ggtcgtaaac
     4081 atccgcgtta tcgttgtcgt agcatgggtt ttccgaaaca ttgtggtaat ggtacagttg
     4141 caatggcaga atgggatgca ttttgcgaag aacaggttct ggatctgctg ggtgatgccg
     4201 aacgtctgga aaaagtttgg gttgcaggta gcgatagcgc agttgaactg gccgaagtta
     4261 atgcggaact ggtcgatctc accagtctga ttggaagtcc cgcatatcgt gcgggtagtc
     4321 ctcagcgtga agcactggat gcacgtattg cagcactggc agcacgtcaa gaagaactgg
     4381 aaggtctgga agcacgtccg agcggttggg aatggcgtga aacaggtcag cgttttggtg
     4441 attggtggcg tgagcaggat accgcagcaa aaaatacctg gctgcgtagt atgaatgttc
     4501 gcctgacctt tgatgttcgc ggtggcctga cccgcaccat tgattttggc gatctgcaag
     4561 aatatgaaca gcatctgcgt ctgggtagcg ttgttgaacg tctgcatacc ggcatgagca
     4621 ccggctgctg tccaggttgt tgcgctgcta acgacgaaaa ctacgctctg gctgcttaag
     4681 aattcgcgcg gccgcggcct aggcggcctc ctgtgtgaaa ttgttatccg ctttaattaa
     4741 aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt
     4801 ttgtcggtga acgctctcct gagtaggaca aatccgccgc cctagacagc tgggcgcgcc
     4861 cccgccgcat ccataccgcc agttgtttac cctcacaacg ttccagtaac cgggcatgtt
     4921 catcatcagt aacccgtatc gtgagcatcc tctctcgttt catcggtatc attaccccca
     4981 tgaacagaaa tcccccttac acggaggcat cagtgaccaa acaggaaaaa accgccctta
     5041 acatggcccg ctttatcaga agccagacat taacgcttct ggagaaactc aacgagctgg
     5101 acgcggatga acaggcagac atctgtgaat cgctgcacga ccacgctgat gagctttacc
     5161 gcagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     5221 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     5281 cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag
     5341 tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg
     5401 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc
     5461 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     5521 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     5581 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5641 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5701 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5761 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5821 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5881 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5941 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     6001 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     6061 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     6121 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     6181 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     6241 tacggggccg gcctacggcc agcctcgcag agcaggattc ccgttgagca ccgccaggtg
     6301 cgaataaggg acagtgaaga aggaacaccc gctcgcgggt gggcctactt cacctatcct
     6361 gcccggctga cgccgttgga tacaccaagg aaagtctaca cgaacccttt ggcaaaatcc
     6421 tgtatatcgt gcgaaaaagg atggatatac cgaaaaaatc gctataatga ccccgaagca
     6481 gggttatgca gcggaaaagg acaacgcgcg gaccgcggtc caattaatta ttagaaaaat
     6541 tcatccagca tcagatgaaa ttgcagtttg ttcatatccg gattatcaat gccatatttc
     6601 tgaaacagac gtttttgcag gctcgggcta aattcgccca ggcagttcca cagaatggcc
     6661 agatcctgat aacgatccgc aatgcccaca cggcccacat caatgcagcc aatcagtttg
     6721 ccttcatcga aaatcaggtt atccaggcta aaatcgccgt gggtcaccac gctatccggg
     6781 ctaaacggca gcagtttatg catttctttc cacacctgtt ccaccggcca gccgttacgt
     6841 tcatcatcaa aatcgctcgc atccaccagg ccgttgttca tacggctctg cgcctgggcc
     6901 agacgaaaca cacgatcgct gttaaacggg cagttgcaca ccggaatgct atgcagacga
     6961 cgcagaaaca cggccagcgc atccacaatg ttttcgccgc tatccggata ttcttccagc
     7021 acctgaaacg cggttttgcc cggaatcgcg gtggtcagca gccacgcatc atccggggtg
     7081 cgaataaaat gtttaatggt cggcagcggc ataaattcgg tcagccagtt cagacgcacc
     7141 atttcatcgg tcacatcgtt cgccacgctg cctttgccat gtttcagaaa cagttccggc
     7201 gcatccggtt tgccatacag acgataaatg gtcgcgccgc tctgacccac gttatcacgc
     7261 gcccatttat agccatacag atccgcatcc atgttgctgt tcagacgcgg acggctacag
     7321 ctcgtttcac gctgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac
     7381 agttttattg ttcatgatga tatattttta tcttgtgcaa tgtaacatca gagattttga
     7441 gacacaaatt taaatcgtaa ttattgggga cccctggatt ctcaccaata aaaaacgccc
     7501 ggcggcaacc gagcgttctg aacaaatcca gatggagttc tgaggtcatt actggatcta
     7561 tcaacaggag tccaag
//
