LOCUS       sequence-307642-        5698 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Derivative of pASPIre2 lacking mCherry but containing a silent 150 
            bp DNA spacer flanked by attB/P sites; main uASPIre plasmid used in 
            this study.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5698)
  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 5698)
  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: pASPIre3
FEATURES             Location/Qualifiers
     source          1..5698
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    27..76
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attB site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
     protein_bind    228..275
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     promoter        356..474
                     /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             2054..2764
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             2054..2764
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(2081..2109)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2704..2724
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             2771..2803
                     /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(2832..2854)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     terminator      2869..2955
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             3135..3326
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3320..3342)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3428..3568
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3483..3502
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3583..3600)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3754..4342)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3834..3853)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     oriT            4378..4486
                     /note="incP origin of transfer"
     CDS             complement(4653..5468)
                     /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     5460..5479
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     terminator      complement(5600..5694)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
ORIGIN
        1 actagtaagc ttcacatctg cagtaatcgg ccggcttgtc gacgacggcg gtctccgtcg
       61 tcaggatcat ccgggcccat ggatcgctta gtcacctttg ggccacggtc cgctacctta
      121 caggaatagt actcgtcctt taatttccct tgcatatatg ttgcgtttct tcgacctagt
      181 actcgctccc ttaggagaaa gacagatagc ttcttacccg ccatgggggt ttgtaccgta
      241 caccactgag accgcggtgg ttgaccagac aaaccacgaa ggttctgtta agtaactgaa
      301 cccaatgtcg ttagtgacgc ttacctctta agaggtcact gacctaacag gatcccacca
      361 caattcagca aattgtgaac atcatcacgt tcatctttcc ctggttgcca atggcccatt
      421 ttcctgtcag taacgagaag gtcgcgaatt caggcgcttt ttagactggt cgtaatgaag
      481 ggtaccataa atgcttcaat aatattgaaa aggattctag atgcgagctc tggttgttat
      541 tcgtctgagc cgtgttaccg atgcaaccac cagtccggaa cgtcagctgg aaagctgtca
      601 gcagctgtgt gcacagcgtg gttgggatgt tgttggtgtt gcagaggatc tggatgttag
      661 cggtgcagtt gatccgtttg atcgtaaacg tcgtccgaat ctggcacgtt ggctggcatt
      721 tgaagaacag ccgtttgatg ttattgttgc ctatcgtgtt gatcgtctga cccgtagcat
      781 tcgtcatctg caacagctgg ttcattgggc agaagatcat aaaaaactgg ttgtgagcgc
      841 aaccgaagca cattttgata ccaccacccc gtttgcagca gttgttattg cactgatggg
      901 caccgttgca cagatggaac tggaagcaat taaagaacgt aatcgtagcg cagcccattt
      961 taacattcgt gcaggtaaat atcgtggtag cctgcctccg tggggttatc tgccgacgcg
     1021 tgtagatggt gaatggcgtc tggttcctga cccggttcag cgtgaacgta ttctggaagt
     1081 atatcatcgt gtggtggata atcatgaacc gctgcatctg gttgcacatg atctgaatcg
     1141 tcgtggtgtt ctgagtccca aagattattt tgctcagctg caaggtcgtg aaccgcaggg
     1201 tcgtgaatgg tctgcaaccg cactgaaacg tagcatgatt agcgaagcaa tgctgggtta
     1261 tgcaaccctg aatggtaaaa ccgttcgtga tgatgatggt gcaccgctgg ttcgtgcaga
     1321 accgattctg acacgtgaac agctggaagc actgcgtgcc gaactggtta aaaccagccg
     1381 tgcaaaaccg gcagttagca ccccgagcct gctgctgcgt gttctgtttt gtgcagtttg
     1441 tggtgaaccg gcatacaaat ttgccggtgg tggtcgtaaa catccgcgtt atcgttgtcg
     1501 tagcatgggt tttccgaaac attgtggtaa tggtacagtt gcaatggcag aatgggatgc
     1561 attttgcgaa gaacaggttc tggatctgct gggtgatgcc gaacgtctgg aaaaagtttg
     1621 ggttgcaggt agcgatagcg cagttgaact ggccgaagtt aatgcggaac tggtcgatct
     1681 caccagtctg attggaagtc ccgcatatcg tgcgggtagt cctcagcgtg aagcactgga
     1741 tgcacgtatt gcagcactgg cagcacgtca agaagaactg gaaggtctgg aagcacgtcc
     1801 gagcggttgg gaatggcgtg aaacaggtca gcgttttggt gattggtggc gtgagcagga
     1861 taccgcagca aaaaatacct ggctgcgtag tatgaatgtt cgcctgacct ttgatgttcg
     1921 cggtggcctg acccgcacca ttgattttgg cgatctgcaa gaatatgaac agcatctgcg
     1981 tctgggtagc gttgttgaac gtctgcatac cggcatgagc accggcggtg gcagcggcgg
     2041 ttctggtggc tctagcaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga
     2101 attagatggt gatgttaatg ggcacaaatt ttctgtccgt ggagagggtg aaggtgatgc
     2161 tacaaacgga aaactcaccc ttaaatttat ttgcactact ggaaaactac ctgttccgtg
     2221 gccaacactt gtcactactc tgacctatgg tgttcaatgc ttttcccgtt atccggatca
     2281 catgaaacgg catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaacgcac
     2341 tatatctttc aaagatgacg ggacctacaa gacgcgtgct gaagtcaagt ttgaaggtga
     2401 tacccttgtt aatcgtatcg agttaaaggg tattgatttt aaagaagatg gaaacattct
     2461 tggacacaaa ctcgagtaca actttaactc acacaatgta tacatcacgg cagacaaaca
     2521 aaagaatgga atcaaagcta acttcaaaat tcgccacaac gttgaagatg gttccgttca
     2581 actagcagac cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga
     2641 caaccattac ctgtcgacac aatctgtcct ttcgaaagat cccaacgaaa agcgtgacca
     2701 catggtcctt cttgagtttg taactgctgc tgggattaca catggcatgg atgaactcta
     2761 caaaaggcct gctgctaacg acgaaaacta cgctctggct gcttaataag cggccgcggc
     2821 ctaggcggcc tcctgtgtga aattgttatc cgctttaatt aaaggcatca aataaaacga
     2881 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc
     2941 ctgagtagga caaatccgcc gccctagaca gctgggcgcg cccccgccgc atccataccg
     3001 ccagttgttt accctcacaa cgttccagta accgggcatg ttcatcatca gtaacccgta
     3061 tcgtgagcat cctctctcgt ttcatcggta tcattacccc catgaacaga aatccccctt
     3121 acacggaggc atcagtgacc aaacaggaaa aaaccgccct taacatggcc cgctttatca
     3181 gaagccagac attaacgctt ctggagaaac tcaacgagct ggacgcggat gaacaggcag
     3241 acatctgtga atcgctgcac gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt
     3301 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     3361 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     3421 gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta
     3481 tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag
     3541 atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct
     3601 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     3661 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     3721 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     3781 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     3841 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     3901 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     3961 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4021 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4081 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4141 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     4201 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     4261 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     4321 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggc cggcctacgg
     4381 ccagcctcgc agagcaggat tcccgttgag caccgccagg tgcgaataag ggacagtgaa
     4441 gaaggaacac ccgctcgcgg gtgggcctac ttcacctatc ctgcccggct gacgccgttg
     4501 gatacaccaa ggaaagtcta cacgaaccct ttggcaaaat cctgtatatc gtgcgaaaaa
     4561 ggatggatat accgaaaaaa tcgctataat gaccccgaag cagggttatg cagcggaaaa
     4621 ggacaacgcg cggaccgcgg tccaattaat tattagaaaa attcatccag catcagatga
     4681 aattgcagtt tgttcatatc cggattatca atgccatatt tctgaaacag acgtttttgc
     4741 aggctcgggc taaattcgcc caggcagttc cacagaatgg ccagatcctg ataacgatcc
     4801 gcaatgccca cacggcccac atcaatgcag ccaatcagtt tgccttcatc gaaaatcagg
     4861 ttatccaggc taaaatcgcc gtgggtcacc acgctatccg ggctaaacgg cagcagttta
     4921 tgcatttctt tccacacctg ttccaccggc cagccgttac gttcatcatc aaaatcgctc
     4981 gcatccacca ggccgttgtt catacggctc tgcgcctggg ccagacgaaa cacacgatcg
     5041 ctgttaaacg ggcagttgca caccggaatg ctatgcagac gacgcagaaa cacggccagc
     5101 gcatccacaa tgttttcgcc gctatccgga tattcttcca gcacctgaaa cgcggttttg
     5161 cccggaatcg cggtggtcag cagccacgca tcatccgggg tgcgaataaa atgtttaatg
     5221 gtcggcagcg gcataaattc ggtcagccag ttcagacgca ccatttcatc ggtcacatcg
     5281 ttcgccacgc tgcctttgcc atgtttcaga aacagttccg gcgcatccgg tttgccatac
     5341 agacgataaa tggtcgcgcc gctctgaccc acgttatcac gcgcccattt atagccatac
     5401 agatccgcat ccatgttgct gttcagacgc ggacggctac agctcgtttc acgctgaata
     5461 tggctcataa caccccttgt attactgttt atgtaagcag acagttttat tgttcatgat
     5521 gatatatttt tatcttgtgc aatgtaacat cagagatttt gagacacaaa tttaaatcgt
     5581 aattattggg gacccctgga ttctcaccaa taaaaaacgc ccggcggcaa ccgagcgttc
     5641 tgaacaaatc cagatggagt tctgaggtca ttactggatc tatcaacagg agtccaag
//
