LOCUS       proinsulin-mGold        4380 bp DNA     circular SYN 16-JAN-2024
DEFINITION  Plasmid for bacterial expression of a proinsulin-mGold fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4380)
  AUTHORS   Glick Lab
  TITLE     Direct Submission
  JOURNAL   Exported May 6, 2024 from SnapGene 7.1.0b
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4380
                     /lab_host="Escherichia coli"
                     /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             115..374
                     /codon_start=1
                     /label=proinsulin
                     /note="Human proinsulin gene w/o introns"
                     /translation="MFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVEL
                     GGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYC"
     CDS             join(376..378,379..381,382..1095)
                     /codon_start=1
                     /product="photostable derivative of mVenus (Lee et al., 
                     2020)"
                     /label=mGold
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTSLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     terminator      1126..1220
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      1992..2078
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_feature    2234..2374
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(2560..3148)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number colE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(3319..4110,4111..4179))
                     /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"
     promoter        complement(4180..4284)
                     /gene="bla"
                     /label=AmpR promoter
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 gaccagcctg ggctacggcc tgcagtgctt cgcccgctac
      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 tatcaccgcc
      841 gacaagcaga agaacggcat caaggccaac ttcaagatcc gccacaacat cgaggacggc
      901 ggcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
      961 ctgcccgaca accactacct gagctaccag 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 tctgtgatgg
     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 gccgctctag agctgcctcg
     2101 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     2161 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     2221 gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct
     2281 taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc
     2341 gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga
     2401 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     2461 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     2521 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     2581 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     2641 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     2701 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     2761 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     2821 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     2881 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     2941 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     3001 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     3061 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     3121 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     3181 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     3241 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     3301 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     3361 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     3421 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     3481 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     3541 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     3601 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     3661 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     3721 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     3781 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     3841 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     3901 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     3961 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     4021 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     4081 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     4141 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     4201 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     4261 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg ctaagaaacc
     4321 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcac
//
