LOCUS       sequence-446162-        4370 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses proinsulin fused to msGFP2 (C48S C70S) in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4370)
  TITLE     Yellow and oxidation-resistant derivatives of a monomeric 
            superfolder GFP
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4370)
  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: 
            "Unpublished"
COMMENT     Sequence Label: proinsulin-msGFP2 (C48S C70S)
FEATURES             Location/Qualifiers
     source          1..4370
                     /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             376..1083
                     /codon_start=1
                     /product="photostable monomeric superfolder GFP (Valbuena 
                     et al., 2020)"
                     /label=msGFP2
                     /translation="MDSTESLFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FISTTGKLPVPWPTLVTTLTYGVQSFSRYPDHMKQHDFFKSAMPEGYVQERTITFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITGGSGGS"
     primer_bind     complement(418..439)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1026..1047
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     terminator      1117..1211
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(1321..1340)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1983..2069
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2116..2138)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2224..2364
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2279..2298
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2379..2396)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2550..3138)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2630..2649)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3309..4100,4101..4169))
                     /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     3932..3951
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4170..4274)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4338..4356
                     /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 gcaccatgga tagcactgag agcctgttca ccggggtggt gcccatcctg
      421 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgcgcggcga gggcgagggc
      481 gatgccacca acggcaagct gaccctgaag ttcatcagca ccaccggcaa gctgcccgtg
      541 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agagcttcag ccgctacccc
      601 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      661 cgcaccatca ccttcaagga cgacggcacc tacaagaccc gcgccgaggt gaagttcgag
      721 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
      781 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat caccgccgac
      841 aagcagaaga acggcatcaa ggccaacttc aagatccgcc acaacgtgga ggacggcagc
      901 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
      961 cccgacaacc actacctgag cacccagtcc aagctgagca aagaccccaa cgagaagcgc
     1021 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactggagg cagtggcggt
     1081 tcttaaagcg gccgcaagct taattagctg agcttggact cctgttgata gatccagtaa
     1141 tgacctcaga actccatctg gatttgttca gaacgctcgg ttgccgccgg gcgtttttta
     1201 ttggtgagaa tccaagctag cttggcgaga ttttcaggag ctaaggaagc taaaatggag
     1261 aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt
     1321 gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg
     1381 gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt
     1441 cttgcccgcc tgatgaatgc tcatccggaa tttcgtatgg caatgaaaga cggtgagctg
     1501 gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt
     1561 tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa
     1621 gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg
     1681 tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat
     1741 atggacaact tcttcgcccc cgttttcacc atgcatgggc aaatattata cgcaaggcga
     1801 caaggtgctg atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg gcttccatgt
     1861 cggcagaatg cttaatgaat tacaacagta ctgcgatgag tggcagggcg gggcgtaatt
     1921 tttttaaggc agttattggt gcccttaaac gcctggggta atgactctct agcttgaggc
     1981 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt
     2041 cggtgaacgc tctcctgagt aggacaaatc cgccctctag agctgcctcg cgcgtttcgg
     2101 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     2161 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     2221 gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg
     2281 gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc
     2341 gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc
     2401 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     2461 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     2521 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     2581 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     2641 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     2701 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     2761 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     2821 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     2881 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     2941 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     3001 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     3061 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     3121 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     3181 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     3241 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     3301 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     3361 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     3421 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     3481 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     3541 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     3601 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     3661 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     3721 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     3781 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     3841 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     3901 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     3961 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     4021 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     4081 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     4141 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     4201 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     4261 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg ctaagaaacc attattatca
     4321 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcac
//
