LOCUS       sequence-403168-        5692 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Derivative of pASPIre3. Contains SpeI restriction site within the 
            CDS of bxb1 to enable diversification of the 5'-UTR and codons 
            2-16..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5692)
  AUTHORS   Hollerer S, Jeschek M
  TITLE     Ultradeep characterisation of translational sequence determinants 
            refutes rare-codon hypothesis and unveils quadruplet base pairing of
            initiator tRNA and transcript.
  JOURNAL   Nucleic Acids Res. 2023 Mar 21;51(5):2377-2396. doi: 
            10.1093/nar/gkad040.
  PUBMED    36727459
REFERENCE   2  (bases 1 to 5692)
  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"; doi: "10"; journalName: 
            "Nucleic Acids Res"; date: "2023-03-21- 21"; volume: "51"; issue: 
            "5"; pages: "2377-2396"
COMMENT     Sequence Label: pASPIre4
FEATURES             Location/Qualifiers
     source          1..5692
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    21..70
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attB site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
     protein_bind    222..269
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     promoter        350..468
                     /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             2048..2758
                     /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             2048..2758
                     /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(2075..2103)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2698..2718
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             2765..2797
                     /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(2826..2848)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     terminator      2863..2949
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             3129..3320
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3314..3336)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3422..3562
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3477..3496
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3577..3594)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3748..4336)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3828..3847)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     oriT            4372..4480
                     /note="incP origin of transfer"
     CDS             complement(4647..5462)
                     /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     5454..5473
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     terminator      complement(5594..5688)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
ORIGIN
        1 aagcttcaca tctgcagtaa tcggccggct tgtcgacgac ggcggtctcc gtcgtcagga
       61 tcatccgggc atcgcttagt cacctttggg ccacggtccg ctaccttaca ggaatagtac
      121 tcgtccttta atttggaatg aaccatggca gtcagttgtg ttgcgtttct tcgacctagt
      181 actcgctccc ttaggagaaa gacagatagc ttcttacccg gggtttgtac cgtacaccac
      241 tgagaccgcg gtggttgacc agacaaacca cgaaggttct gttaagtaac tgaacccaat
      301 gtcgttagtg acgcttacct cttaagaggt cactgaccta acaggatccc accacaattc
      361 agcaaattgt gaacatcatc acgttcatct ttccctggtt gccaatggcc cattttcctg
      421 tcagtaacga gaaggtcgcg aattcaggcg ctttttagac tggtcgtaat gaagagctca
      481 ataaatattt aatttatctc agaaaggcta agacatgcga gcactggttg ttattcgtct
      541 gagccgtgtt accgatgcaa ccactagtcc ggaacgtcag ctggaaagct gtcagcagct
      601 gtgtgcacag cgtggttggg atgttgttgg tgttgcagag gatctggatg ttagcggtgc
      661 agttgatccg tttgatcgta aacgtcgtcc gaatctggca cgttggctgg catttgaaga
      721 acagccgttt gatgttattg ttgcctatcg tgttgatcgt ctgacccgta gcattcgtca
      781 tctgcaacag ctggttcatt gggcagaaga tcataaaaaa ctggttgtga gcgcaaccga
      841 agcacatttt gataccacca ccccgtttgc agcagttgtt attgcactga tgggcaccgt
      901 tgcacagatg gaactggaag caattaaaga acgtaatcgt agcgcagccc attttaacat
      961 tcgtgcaggt aaatatcgtg gtagcctgcc tccgtggggt tatctgccga cgcgtgtaga
     1021 tggtgaatgg cgtctggttc ctgacccggt tcagcgtgaa cgtattctgg aagtatatca
     1081 tcgtgtggtg gataatcatg aaccgctgca tctggttgca catgatctga atcgtcgtgg
     1141 tgttctgagt cccaaagatt attttgctca gctgcaaggt cgtgaaccgc agggtcgtga
     1201 atggtctgca accgcactga aacgtagcat gattagcgaa gcaatgctgg gttatgcaac
     1261 cctgaatggt aaaaccgttc gtgatgatga tggtgcaccg ctggttcgtg cagaaccgat
     1321 tctgacacgt gaacagctgg aagcactgcg tgccgaactg gttaaaacca gccgtgcaaa
     1381 accggcagtt agcaccccga gcctgctgct gcgtgttctg ttttgtgcag tttgtggtga
     1441 accggcatac aaatttgccg gtggtggtcg taaacatccg cgttatcgtt gtcgtagcat
     1501 gggttttccg aaacattgtg gtaatggtac agttgcaatg gcagaatggg atgcattttg
     1561 cgaagaacag gttctggatc tgctgggtga tgccgaacgt ctggaaaaag tttgggttgc
     1621 aggtagcgat agcgcagttg aactggccga agttaatgcg gaactggtcg atctcaccag
     1681 tctgattgga agtcccgcat atcgtgcggg tagtcctcag cgtgaagcac tggatgcacg
     1741 tattgcagca ctggcagcac gtcaagaaga actggaaggt ctggaagcac gtccgagcgg
     1801 ttgggaatgg cgtgaaacag gtcagcgttt tggtgattgg tggcgtgagc aggataccgc
     1861 agcaaaaaat acctggctgc gtagtatgaa tgttcgcctg acctttgatg ttcgcggtgg
     1921 cctgacccgc accattgatt ttggcgatct gcaagaatat gaacagcatc tgcgtctggg
     1981 tagcgttgtt gaacgtctgc ataccggcat gagcaccggc ggtggcagcg gcggttctgg
     2041 tggctctagc aaaggagaag aacttttcac tggagttgtc ccaattcttg ttgaattaga
     2101 tggtgatgtt aatgggcaca aattttctgt ccgtggagag ggtgaaggtg atgctacaaa
     2161 cggaaaactc acccttaaat ttatttgcac tactggaaaa ctacctgttc cgtggccaac
     2221 acttgtcact actctgacct atggtgttca atgcttttcc cgttatccgg atcacatgaa
     2281 acggcatgac tttttcaaga gtgccatgcc cgaaggttat gtacaggaac gcactatatc
     2341 tttcaaagat gacgggacct acaagacgcg tgctgaagtc aagtttgaag gtgataccct
     2401 tgttaatcgt atcgagttaa agggtattga ttttaaagaa gatggaaaca ttcttggaca
     2461 caaactcgag tacaacttta actcacacaa tgtatacatc acggcagaca aacaaaagaa
     2521 tggaatcaaa gctaacttca aaattcgcca caacgttgaa gatggttccg ttcaactagc
     2581 agaccattat caacaaaata ctccaattgg cgatggccct gtccttttac cagacaacca
     2641 ttacctgtcg acacaatctg tcctttcgaa agatcccaac gaaaagcgtg accacatggt
     2701 ccttcttgag tttgtaactg ctgctgggat tacacatggc atggatgaac tctacaaaag
     2761 gcctgctgct aacgacgaaa actacgctct ggctgcttaa taagcggccg cggcctaggc
     2821 ggcctcctgt gtgaaattgt tatccgcttt aattaaaggc atcaaataaa acgaaaggct
     2881 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
     2941 aggacaaatc cgccgcccta gacagctggg cgcgcccccg ccgcatccat accgccagtt
     3001 gtttaccctc acaacgttcc agtaaccggg catgttcatc atcagtaacc cgtatcgtga
     3061 gcatcctctc tcgtttcatc ggtatcatta cccccatgaa cagaaatccc ccttacacgg
     3121 aggcatcagt gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc
     3181 agacattaac gcttctggag aaactcaacg agctggacgc ggatgaacag gcagacatct
     3241 gtgaatcgct gcacgaccac gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg
     3301 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     3361 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     3421 gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc
     3481 atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt
     3541 aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     3601 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3661 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3721 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3781 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3841 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     3901 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     3961 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4021 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4081 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4141 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
     4201 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4261 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4321 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggccggcct acggccagcc
     4381 tcgcagagca ggattcccgt tgagcaccgc caggtgcgaa taagggacag tgaagaagga
     4441 acacccgctc gcgggtgggc ctacttcacc tatcctgccc ggctgacgcc gttggataca
     4501 ccaaggaaag tctacacgaa ccctttggca aaatcctgta tatcgtgcga aaaaggatgg
     4561 atataccgaa aaaatcgcta taatgacccc gaagcagggt tatgcagcgg aaaaggacaa
     4621 cgcgcggacc gcggtccaat taattattag aaaaattcat ccagcatcag atgaaattgc
     4681 agtttgttca tatccggatt atcaatgcca tatttctgaa acagacgttt ttgcaggctc
     4741 gggctaaatt cgcccaggca gttccacaga atggccagat cctgataacg atccgcaatg
     4801 cccacacggc ccacatcaat gcagccaatc agtttgcctt catcgaaaat caggttatcc
     4861 aggctaaaat cgccgtgggt caccacgcta tccgggctaa acggcagcag tttatgcatt
     4921 tctttccaca cctgttccac cggccagccg ttacgttcat catcaaaatc gctcgcatcc
     4981 accaggccgt tgttcatacg gctctgcgcc tgggccagac gaaacacacg atcgctgtta
     5041 aacgggcagt tgcacaccgg aatgctatgc agacgacgca gaaacacggc cagcgcatcc
     5101 acaatgtttt cgccgctatc cggatattct tccagcacct gaaacgcggt tttgcccgga
     5161 atcgcggtgg tcagcagcca cgcatcatcc ggggtgcgaa taaaatgttt aatggtcggc
     5221 agcggcataa attcggtcag ccagttcaga cgcaccattt catcggtcac atcgttcgcc
     5281 acgctgcctt tgccatgttt cagaaacagt tccggcgcat ccggtttgcc atacagacga
     5341 taaatggtcg cgccgctctg acccacgtta tcacgcgccc atttatagcc atacagatcc
     5401 gcatccatgt tgctgttcag acgcggacgg ctacagctcg tttcacgctg aatatggctc
     5461 ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca tgatgatata
     5521 tttttatctt gtgcaatgta acatcagaga ttttgagaca caaatttaaa tcgtaattat
     5581 tggggacccc tggattctca ccaataaaaa acgcccggcg gcaaccgagc gttctgaaca
     5641 aatccagatg gagttctgag gtcattactg gatctatcaa caggagtcca ag
//
