LOCUS       sequence-236009-        6540 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  medium stringency SE-PACE AP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6540)
  AUTHORS   Wang T, Badran AH, Huang TP, Liu DR
  TITLE     Continuous directed evolution of proteins with improved soluble 
            expression.
  JOURNAL   Nat Chem Biol. 2018 Aug 20. pii: 10.1038/s41589-018-0121-5. doi: 
            10.1038/s41589-018-0121-5.
  PUBMED    30127387
REFERENCE   2  (bases 1 to 6540)
  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 Chem 
            Biol. 2018 Aug 20. pii: 10.1038/s41589-018-0121-5. doi: 
            10.1038/s41589-018-0121-5."
COMMENT     Sequence Label: pTW051b4
FEATURES             Location/Qualifiers
     source          1..6540
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1..792)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(793..884)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      893..1481
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1382..1401
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1635..1652
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1667..1807
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1733..1752)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1893..1915
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1909..2100)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     terminator      complement(6120..6147)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(6239..6325)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     6478..6495
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
ORIGIN
        1 ttatttgccg actaccttgg tgatctcgcc tttcacgtag tggacaaatt cttccaactg
       61 atctgcgcgc gaggccaagc gatcttcttc ttgtccaaga taagcctgtc tagcttcaag
      121 tatgacgggc tgatactggg ccggcaggcg ctccattgcc cagtcggcag cgacatcctt
      181 cggcgcgatt ttgccggtta ctgcgctgta ccaaatgcgg gacaacgtaa gcactacatt
      241 tcgctcatct ccagcccagt cgggcggcga gttccatagc gttaaggttt catttagcgc
      301 ctcaaataga tcctgttcag gaaccggatc aaagagttcc tccgccgctg gacctaccaa
      361 ggcaacgcta tgttctcttg cttttgtcag caagatagcc agatcaatgt cgatcgtggc
      421 tggctcgaag atacctgcaa gaatgtcatt gcgctgccat tctccaaatt gcagttcgcg
      481 cttagctgga taacgccacg gaatgatgtc gtcgtgcaca acaatggtga cttctacagc
      541 gcggagaatc tcgctctctc caggggaagc cgaagtttcc aaaaggtcgt tgatcaaagc
      601 tcgccgcgtt gtttcatcaa gccttacggt caccgtaacc agcaaatcaa tatcactgtg
      661 tggcttcagg ccgccatcca ctgcggagcc gtacaaatgt acggccagca acgtcggttc
      721 gagatggcgc tcgatgacgc caactacctc tgatagttga gtcgatactt cggcgatcac
      781 cgcttccctc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
      841 catgagcgga tacatatttg aatgtattta gaaaaatagg ccaaataggc cgttgagatc
      901 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
      961 tttgtttgcc ggatcaagag ctaccacctc tttttccgaa ggtaactggc ttcagcagag
     1021 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     1081 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     1141 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     1201 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     1261 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     1321 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     1381 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     1441 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     1501 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     1561 ctgattctgt ggataaccgt attacagcct ttgagtgagc tgataccgct cgccgcagcc
     1621 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     1681 ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct
     1741 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     1801 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtcttctccc
     1861 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     1921 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     1981 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     2041 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     2101 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     2161 gaggatgctc acgatacggg ttactgatga tgaacaagag gacatccggt caaataaaac
     2221 gaaaggctca gtcgaaagac tgggcctttc gttttgctga ggagacttag ggaccctact
     2281 aatacgactc cctataggga gaaagacctg caggtgcagt aaaaaaggaa aaaaatgatc
     2341 aaaattgcca cgcgtaaata tttaggcaaa cagaatgttt atgatatcgg tgtcgagcgc
     2401 gatcataatt tcgcgctgaa aaacggcttt atcgccagca attgttttaa tgttgttcct
     2461 ttctattctc actccgctga aactgttgaa agttgtttag caaaacccca tacagaaaat
     2521 tcatttacta acgtctggaa agacgacaaa actttagatc gttacgctaa ctatgagggc
     2581 tgtctgtgga atgctacagg cgttgtagtt tgtactggtg acgaaactca gtgttacggt
     2641 acatgggttc ctattgggct tgctatccct gaaaatgagg gtggtggctc tgagggtggc
     2701 ggttctgagg gtggcggttc tgagggtggc ggtactaaac ctcctgagta cggtgataca
     2761 cctattccgg gctatactta tatcaaccct ctcgacggca cttatccgcc tggtactgag
     2821 caaaaccccg ctaatcctaa tccttctctt gaggagtctc agcctcttaa tactttcatg
     2881 tttcagaata ataggttccg aaataggcag ggggcattaa ctgtttatac gggcactgtt
     2941 actcaaggca ctgaccccgt taaaacttat taccagtaca ctcctgtatc atcaaaagcc
     3001 atgtatgacg cttactggaa cggtaaattc agagactgcg ctttccattc tggctttaat
     3061 gaggatccat tcgtttgtga atatcaaggc caatcgtctg acctgcctca acctcctgtc
     3121 aatgctggcg gcggctctgg tggtggttct ggtggcggct ctgagggtgg tggctctgag
     3181 ggtggcggtt ctgagggtgg cggctctgag ggaggcggtt ccggtggtgg ctctggttcc
     3241 ggtgattttg attatgaaaa gatggcaaac gctaataagg gggctatgac cgaaaatgcc
     3301 gatgaaaacg cgctacagtc tgacgctaaa ggcaaacttg attctgtcgc tactgattac
     3361 ggtgctgcta tcgatggttt cattggtgac gtttccggcc ttgctaatgg taatggtgct
     3421 actggtgatt ttgctggctc taattcccaa atggctcaag tcggtgacgg tgataattca
     3481 cctttaatga ataatttccg tcaatattta ccttccctcc ctcaatcggt tgaatgtcgc
     3541 ccttttgtct ttggcgctgg taaaccttac gagttcagta tcgactgcga taagatcaac
     3601 ctgttccgcg gtgtctttgc gtttctttta tatgttgcca cctttatgta tgtattttct
     3661 acgtttgcta acatactgcg taataaggag tcttaaacgg agccaatgta cgcaaaaaac
     3721 cccgcttcag cggggttttt tcgccaaaaa aaaccccgcc ctgtcagggg cggggttttt
     3781 ttttagggcc ctactgactg ttacgcgaac gcgaagtccg actctaagat gtcacggagg
     3841 ttcaagttac ctttagccgg aagtgctggc attttgtcca attgagactc gtgcaactgg
     3901 tcagcgaact ggtcgtagaa atcagccagt acatcacaag actcatatgt gtcaaccata
     3961 gtttcgcgca ctgctttgaa caggttcgca gcgtcagccg gaatggtacc gaaggagtcg
     4021 tgaatcagtg caaaagattc gattccgtac ttctcgtgtg cccacactac agtcttacga
     4081 aggtggctac cgtcttggct gtgtacaaag ttaggagcga taccagactc ctgtttgtgt
     4141 gcatcaatct cgctatcttt gttggtgtta atggtaggct gtaagcggaa ctgaccgagg
     4201 aacatcaggt tcaagcgcgt ctgaataggc ttcttgtatt cctgccacac agggaaacca
     4261 tcaggagtta cccaatgcac agcgcaacgc ttgcgaagaa tctctccagt cttcttatct
     4321 ttgacctcag cagccagcag cttagcagca gacttaagcc agttcattgc ttcaaccgca
     4381 gctaccaccg tcacgctcac agattcccaa atcagcttag ccatgtatcc agcagcctga
     4441 ttcggctgag tgaacatcag acccttgccg gaatcaatag ctggctgaat ggtatcttcc
     4501 agcacttgtt gacggaagcc gaactctttg gacccgtaag ccagcgtcat gactgaactc
     4561 ttagtcacac tgcgagtaac accgtaagcc agccattgac cagccagtgc cttagtgccc
     4621 agcttgactt tctcagagat ttcaccagtg ttctcatcgg tcacggtaac tacttcgtta
     4681 tcggtcccat tgattgcgtc tgcttgtaga atctcgttga ctttcttagc aacaatcccg
     4741 tagatgtcct gaacggtttc actaggaagc aagttaaccg cgcgaccacc tacctcatct
     4801 cggagcatcg cggagaagtg ctggatgcca gagcaagacc cgtcaaacgc cagcggaagg
     4861 gagcagttat agctcaggcc gtggtgctgt accccagcgt actcaaagca gaacgcaagg
     4921 aagcagaacg gagaatcttg ctcagcccac caagtgttct ccagtggaga cttagcgcaa
     4981 gccatgatgt tctcgtggtt ttcctcaatg aacttgatgc gctcagggaa cggaacctta
     5041 tcgacacccg cacagtttgc accgtggatt ttcagccagt agtaaccttc cttaccgatt
     5101 ggtttacctt tcgccagcgt aagcagtcct ttggtcatat cgttaccttg cgggttgaac
     5161 attgacacag cgtaaacacg accgcgccag tccatgttgt aagggaacca gatggcctta
     5221 tggttagcaa acttattggc ttgctcaagc atgaactcaa ggctgatacg gcgagacttg
     5281 cgagccttgt ccttgcggta cacagcagcg gcagcacgtt tccacgcggt gagagcctca
     5341 ggattcatgt cgatgtcttc cggtttcatc gggagttctt cacgctcaat cgcagggatg
     5401 tcctcgaccg gacaatgctt ccacttggtg attacgttgg cgaccgctag gactttcttg
     5461 ttgattttcc atgcggtgtt ttgcgcaatg ttaatcgctt tgtacacctc aggcatgtaa
     5521 acgtcttcgt agcgcatcag tgctttctta ctgtgagtac gcaccagcgc cagaggacga
     5581 cgaccgttag cccaatagcc accaccagta atgccagtcc acggcttagg aggaactacg
     5641 caaggttgga acatcggaga gatgccagcc agcgcacctg cacgggttgc gatagcctca
     5701 gcgtattcag gtgcgagttc gatagtctca gagtcttgac ctactacgcc agcattttgg
     5761 cggtgtaagc taaccattcc ggttgactca atgagcatct cgatgcagcg tactcctaca
     5821 tgaatagagt cttccttatg ccacgaagac cacgcctcgc caccgagtag acccttagag
     5881 agcatgtcag cctcgacaac ttgcataaat gctttcattt ttttttcctc cttactctag
     5941 taaaagttaa acaaaattat ttgtagaggg aaaccgttgt ggtctccctg aatatagata
     6001 ccgagcctta tgcatgcccg taaagttatc cagcaaccac tcatagacct agggcagcag
     6061 atagggacga cgtggtgtta gctgtgctct agaatcctca gcgagtttgt agaaacgcaa
     6121 aaaggccatc cgtcaggatg gccttctgct taatttgatg cctggcagtt tatggcgggc
     6181 gtcctgcccg ccaccctccg ggccgttgct tcgcaacgtt caaatccgct cccggcggat
     6241 ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt
     6301 tcgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc atggggagac
     6361 cccacactac catcggcgct acggcgtttc acttctgagt tcggcatggg gtcaggtggg
     6421 accaccgcgc tactgccgcc aggcaaattc tgttttatca gaccgcttct gcgttctgat
     6481 ttaatctgta tcaggctgaa aatcttctct catccgccaa aacagccatg cccatggcgt
//
