LOCUS       sequence-326358-        4379 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a fusion of human proinsulin and the monomeric enhanced 
            EGFP. This construct can be used as a negative control for 
            superfolder activity in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4379)
  AUTHORS   Valbuena FM, Fizgerald I, Strack RL, Andruska N, Smith L, Glick BS
  TITLE     A photostable monomeric superfolder GFP.
  JOURNAL   Traffic. 2020 May 15. doi: 10.1111/tra.12737.
  PUBMED    32415747
REFERENCE   2  (bases 1 to 4379)
  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: "Traffic. 
            2020 May 15. doi: 10.1111/tra.12737."
COMMENT     Sequence Label: pQE-60NA-proinsulin-mEGFP
FEATURES             Location/Qualifiers
     source          1..4379
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..24,25..30,31..47,48..54)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    30..46
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     protein_bind    62..78
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             97..108
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(376..378,379..381,382..1095)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(421..442)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(682..701)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1029..1050
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     terminator      1126..1220
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(1330..1349)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1992..2078
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2125..2147)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2233..2373
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2288..2307
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2388..2405)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2559..3147)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2639..2658)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3318..4109,4110..4178))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3941..3960
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4179..4283)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4347..4365
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca
       61 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aaccatgttt
      121 gtgaaccaac acctgtgcgg ctcacacctg gtggaagctc tctacctagt gtgcggggaa
      181 cgaggcttct tctacacacc caagacccgc cgggaggcag aggacctgca ggtggggcag
      241 gtggagctgg gcgggggccc tggtgcaggc agcctgcagc ccttggccct ggaggggtcc
      301 ctgcagaagc gtggcattgt ggaacaatgc tgtaccagca tctgctccct ctaccagctg
      361 gagaactact gcaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
      421 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
      481 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
      541 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
      601 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
      661 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
      721 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
      781 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
      841 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
      901 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
      961 ctgcccgaca accactacct gagcacccag tccaagctga gcaaagaccc caacgagaag
     1021 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     1081 gagctgtaca agtaaagcgg ccgcaagctt aattagctga gcttggactc ctgttgatag
     1141 atccagtaat gacctcagaa ctccatctgg atttgttcag aacgctcggt tgccgccggg
     1201 cgttttttat tggtgagaat ccaagctagc ttggcgagat tttcaggagc taaggaagct
     1261 aaaatggaga aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa
     1321 gaacattttg aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg
     1381 gatattacgg cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt
     1441 attcacattc ttgcccgcct gatgaatgct catccggaat ttcgtatggc aatgaaagac
     1501 ggtgagctgg tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact
     1561 gaaacgtttt catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata
     1621 tattcgcaag atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt
     1681 gagaatatgt ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac
     1741 gtggccaata tggacaactt cttcgccccc gttttcacca tgcatgggca aatattatac
     1801 gcaaggcgac aaggtgctga tgccgctggc gattcaggtt catcatgccg tttgtgatgg
     1861 cttccatgtc ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt ggcagggcgg
     1921 ggcgtaattt ttttaaggca gttattggtg cccttaaacg cctggggtaa tgactctcta
     1981 gcttgaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
     2041 gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccctctaga gctgcctcgc
     2101 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     2161 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     2221 cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt
     2281 aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg
     2341 cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac
     2401 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     2461 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     2521 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     2581 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     2641 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     2701 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     2761 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     2821 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     2881 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     2941 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     3001 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     3061 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     3121 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     3181 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     3241 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     3301 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     3361 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     3421 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     3481 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     3541 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     3601 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     3661 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     3721 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     3781 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     3841 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     3901 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     3961 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     4021 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     4081 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     4141 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     4201 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     4261 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctgc taagaaacca
     4321 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcttcac
//
