LOCUS       sequence-271201-        3844 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Low phosphate inducible circular permutation of GFP, which is a 
            substrate of the lon protease.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3844)
  AUTHORS   Menacho-Melgar R, Ye Z, Moreb EA, Yang T, Efromson JP, Decker JS, 
            Wang R, Lynch MD
  TITLE     Scalable, two-stage, autoinduction of recombinant protein expression
            in E. coli utilizing phosphate depletion.
  JOURNAL   Biotechnol Bioeng. 2020 May 22. doi: 10.1002/bit.27440.
  PUBMED    32441815
REFERENCE   2  (bases 1 to 3844)
  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: "Biotechnol 
            Bioeng. 2020 May 22. doi: 10.1002/bit.27440."
COMMENT     Sequence Label: pTKhc-yibDp-GFP-beta-20cp6
FEATURES             Location/Qualifiers
     source          1..3844
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      164..235
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      251..278
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     RBS             571..593
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             673..690
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             694..717
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     terminator      1554..1600
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1748..1764)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1748..1764)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1761..1783)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(1936..2524)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2016..2035)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2660..3475)
                     /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"
     CDS             complement(2660..3475)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /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     3383..3402
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3467..3486
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     3596..3612
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3596..3612
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     terminator      3613..3659
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3773..3795
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3787..3804
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3788..3804
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 tccgcctgac ctatatcaag cttcgtccat tgcatgaggc tcgtcctgaa tgatatcaag
       61 cttcgtccat tgcatgaggc tcgtcctgaa tgatatcaag cttcgtccat tgcatgaggc
      121 tcgtcctgaa tgatatcaag cttgaattcg tttgcccagg catcaaataa aacgaaaggc
      181 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctctacta
      241 gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tatacacagc taacaccacg
      301 tcgtccctat ctgctgccct aggtctatga gtggttgctg gataacgtgc gtaattgtgc
      361 tgatctctta tatagctgct ctcattatct ctctaccctg aagtgactct ctcacctgta
      421 aaaataatat ctcacaggct taatagtttc ttaatacaaa gcctgtaaaa cgtcaggata
      481 acttctatat tcagggagac cacaacggtt tccctctaca aataattttg tttaactttg
      541 agaccacaac ggtttcccac tagaaataat tttgtttaac tttaagaagg agatatacat
      601 atgggccagc tgcgttctct gaacggcgag tggcgctttg cttggttccc tgcgccagag
      661 gctgtcagca gccatcacca tcatcaccac agcctggaag ttctgttcca ggggcccgga
      721 tccatgaaca gccacaatgt ttacatcacc gccgataaac aaaaaaatgg cattaaagcg
      781 aattttaaaa ttcgccacaa cgtggaggat ggcagcgtgc agctggctga tcactaccag
      841 caaaacactc caatcggtga tggtcctgtt ctgctgccag acaatcacta tctgagcacg
      901 caaagcgttc tgtctaaaga tccgaacgag aaacgcgatc atatggttct gctggagttc
      961 gtaaccgcag cgggcatcac gcatggtatg gatgaactgt acaaaggtgg taccggtggt
     1021 tctatgcgta aaggcgaaga gctgttcact ggtgtcgtcc ctattctggt ggaactggat
     1081 ggtgatgtca acggtcataa gttttccgtg cgtggcgagg gtgaaggtga cgcaactaat
     1141 ggtaaactga cgctgaagtt catctgtact actggtaaac tgccggtacc ttggccgact
     1201 ctggtaacga cgctgactta tggtgttcag tgctttgctc gttatccgga ccatatgaag
     1261 cagcatgact tcttcaagtc cgccatgccg gaaggctatg tgcaggaacg cacgatttcc
     1321 tttaaggatg acggcacgta caaaacgcgt gcggaagtga aatttgaagg cgataccctg
     1381 gtaaaccgca ttgagctgaa aggcattgac tttaaagaag acggcaatat cctgggccat
     1441 aagctggaat acaatctcga gtaactcgag tctggtaaaa ctagcattcg acctagcata
     1501 accccgcggg gcctcttcgg gggtctcgcg gggttttttg ctgaaagaag cttcaaataa
     1561 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcgctgcggc
     1621 cgggtcaggt atgatttaaa tggtcagtaa cgggtcttga ggggtttttt gcaatgggtt
     1681 catcccgtgg gaggctaggt ggaggctcag tgatgataag tctgcgatgg tggatgcatg
     1741 tgtcatggtc atagctgttt cctgtgtgaa attgttatcc gctcagaggg cacaatccta
     1801 ttccgcgcta tccgacaatc tccaagacat taggtggagt tcagttcggc gtatggcata
     1861 tgtcgctgga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     1921 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     1981 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     2041 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     2101 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     2161 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     2221 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     2281 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     2341 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     2401 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     2461 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     2521 tcaagaagat cctttgatct tttctacggg gtctgacgct ctattcaaca aagccgccgt
     2581 cccgtcaagt cagcgtaaat gggtaggggg cttcaaatcg tccgctctgc cagtgttaca
     2641 accaattaac aaattctgat tagaaaaact catcgagcat caaatgaaac tgcaatttat
     2701 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     2761 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     2821 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     2881 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagcttatgc atttctttcc
     2941 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     3001 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
     3061 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     3121 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     3181 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     3241 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     3301 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     3361 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     3421 tgttggaatt taatcgcggc ctcgagcaag acgtttcccg ttgaatatgg ctcataacac
     3481 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgatgat atatttttat
     3541 cttgtgcaat gtaacatcag agattttgag acacaacgtg gctttccccc gccgctctag
     3601 aactagtgga tccaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc
     3661 tgttgtttgt cgcattatac gagacgtcca ggttgggata cctgaaacaa aacccatcgt
     3721 acggccaagg aagtctccaa taactgtgat ccaccacaag cgccagggtt ttcccagtca
     3781 cgacgttgta aaacgacggc cagtcatgca taatccgcac gcatctggaa taaggaagtg
     3841 ccat
//
