LOCUS       sequence-147735-        3216 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Supplemental Figure 4. No Promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3216)
  AUTHORS   Perez-Pinera P, Han N, Cleto S, Cao J, Purcell O, Shah KA, Lee K, 
            Ram R, Lu TK
  TITLE     Synthetic biology and microbioreactor platforms for programmable 
            production of biologics at the point-of-care.
  JOURNAL   Nat Commun. 2016 Jul 29;7:12211. doi: 10.1038/ncomms12211.
  PUBMED    27470089
REFERENCE   2  (bases 1 to 3216)
  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 
            Commun."; date: "2016-07-29"; volume: "7:12211. doi"; pages: " 
            10.1038/ncomms12211"
COMMENT     Sequence Label: PP93
FEATURES             Location/Qualifiers
     source          1..3216
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             57..431
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     terminator      497..744
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(513..531)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(819..1407)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(899..918)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    1527..1576
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attB site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
     CDS             join(1700..1702,1703..1705,1706..2416)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1700..1702,1703..1705,1706..2416)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1745..1766)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2006..2025)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2353..2374
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2441..2470
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2486..2503
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2527..2773
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2788..3199
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        join(3207..3216,1..38)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
ORIGIN
        1 aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac taaaccatgg
       61 ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt
      121 tctggaccga ccggctcggg ttctcccggg acttcgtgga ggacgacttc gccggtgtgg
      181 tccgggacga cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg ccggacaaca
      241 ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg
      301 tgtccacgaa cttccgggac gcctccgggc cggccatgac cgagatcggc gagcagccgt
      361 gggggcggga gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg
      421 agcaggactg acacgtccga cggcggccca cgggtcccag gcctcggaga tccgtccccc
      481 ttttcctttg tcgatatcat gtaattagtt atgtcacgct tacattcacg ccctcccccc
      541 acatccgctc taaccgaaaa ggaaggagtt agacaacctg aagtctaggt ccctatttat
      601 ttttttatag ttatgttagt attaagaacg ttatttatat ttcaaatttt tctttttttt
      661 ctgtacagac gcgtgtacgc atgtaacatt atactgaaaa ccttgcttga gaaggttttg
      721 ggacgctcga aggctttaat ttgcaagctg gagaccaaca tgtgagcaaa aggccagcaa
      781 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
      841 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
      901 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
      961 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     1021 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     1081 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     1141 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     1201 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     1261 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     1321 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     1381 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     1441 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagatcaga tctgagctcg
     1501 tttggccgtg gccgtgctcg tcctcgtcgg ccggcttgtc gacgacggcg gtctccgtcg
     1561 tcaggatcat ccgggccaca agcttgctga cagaagcctc aagaaaaaaa aaattcttct
     1621 tcgactatgc tggaggcaga gatgatcgag ccggtagtta actatatata gctaaattgg
     1681 ttccatcacc tcgaaattca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     1741 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     1801 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     1861 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     1921 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     1981 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     2041 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     2101 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     2161 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     2221 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     2281 gctgctgccc gacaaccact acctgagcac ccagagcgcc ctgagcaaag accccaacga
     2341 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     2401 ggacgagctg tacaaggccg cggcggccgc cagctttcta gaacaaaaac tcatctcaga
     2461 agaggatctg aatagcgccg tcgaccatca tcatcatcat cattgaccgg tcttgctaga
     2521 ttctaatcaa gaggatgtca gaatgccatt tgcctgagag atgcaggctt catttttgat
     2581 acttttttat ttgtaaccta tatagtatag gatttttttt gtcattttgt ttcttctcgt
     2641 acgagcttgc tcctgatcag cctatctcgc agctgatgaa tatcttgtgg taggggtttg
     2701 ggaaaatcat tcgagtttga tgtttttctt ggtatttccc actcctcttc agagtacaga
     2761 agattaagtg agaccttcgt ttgtgcggat cccccacaca ccatagcttc aaaatgtttc
     2821 tactcctttt ttactcttcc agattttctc ggactccgcg catcgccgta ccacttcaaa
     2881 acacccaagc acagcatact aaattttccc tctttcttcc tctagggtgt cgttaattac
     2941 ccgtactaaa ggtttggaaa agaaaaaaga gaccgcctcg tttctttttc ttcgtcgaaa
     3001 aaggcaataa aaatttttat cacgtttctt tttcttgaaa tttttttttt tagttttttt
     3061 ctctttcagt gacctccatt gatatttaag ttaataaacg gtcttcaatt tctcaagttt
     3121 cagtttcatt tttcttgttc tattacaact ttttttactt cttgttcatt agaaagaaag
     3181 catagcaatc taatctaagg ggcggtgttg acaatt
//
