LOCUS       sequence-405975-        3599 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Design opt.b (parallel tempering optimization without distance 
            constraints) in modified pSTC0 vector in which kan resistance 
            cassette is replaced with zeo resistance cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3599)
  AUTHORS   Fram B, Su Y, Truebridge I, Riesselman AJ, Ingraham JB, Passera A, 
            Napier E, Thadani NN, Lim S, Roberts K, Kaur G, Stiffler MA, Marks 
            DS, Bahl CD, Khan AR, Sander C, Gauthier NP
  TITLE     Simultaneous enhancement of multiple functional properties using 
            evolution-informed protein design.
  JOURNAL   Nat Commun. 2024 Jun 20;15(1):5141. doi: 10.1038/s41467-024-49119-x.
  PUBMED    38902262
REFERENCE   2  (bases 1 to 3599)
  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: "2024-06-20"; volume: "15(1):5141. doi"; pages: " 
            10.1038/s41467-024-49119-x"
COMMENT     Sequence Label: opt.b_pSTC0-zeo
FEATURES             Location/Qualifiers
     source          1..3599
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(612..986)
                     /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"
     promoter        complement(2002..2106)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2174..2192
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(2196..2239)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     CDS             2266..2973
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="KGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFI
                     CTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNY
                     KTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNF
                     KIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFV
                     TAAGITHGMDELYK"
     primer_bind     complement(2290..2318)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2913..2933
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2996..3067
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3083..3110
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    3117..3137
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      3138..3195
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     primer_bind     complement(3219..3236)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(3390..3599,1..379))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3470..3489)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
       61 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
      121 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
      181 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
      241 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
      301 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
      361 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
      421 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
      481 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
      541 gtctgacagc tcgagtcccg tcaagtcagc gtaatgctct gccagtgtta caaccaatta
      601 accaattctg atcagtcctg ctcctcggcc acgaagtgca cgcagttgcc ggccgggtcg
      661 cgcagggcga actcccgccc ccacggctgc tcgccgatct cggtcatggc cggcccggag
      721 gcgtcccgga agttcgtgga cacgacctcc gaccactcgg cgtacagctc gtccaggccg
      781 cgcacccaca cccaggccag ggtgttgtcc ggcaccacct ggtcctggac cgcgctgatg
      841 aacagggtca cgtcgtcccg gaccacaccg gcgaagtcgt cctccacgaa gtcccgggag
      901 aacccgagcc ggtcggtcca gaactcgacc gctccggcga cgtcgcgcgc ggtgagcacc
      961 ggaacggcac tggtcaactt ggccataaca ccccttgtat tactgtttat gtaagcagac
     1021 agttttattg ttcatgatga tatattttta tcttgtgcaa tgtaacatca gagattttga
     1081 gacacaacgt ggctttgttg aataaatcga acttttgctg agttgaagga tcagctcaag
     1141 ttaccaatgc ttaactgcga cggctgcagc ctcagcgatt aaagcatcgt catattctgc
     1201 gtcctcggtg tcgcgactac tcatcacggc caacactata ggagctccgt ccggtggcca
     1261 cagaactgca atgtcgttgc gagtcccacg gccaccggct ccagttttat cgccaacctc
     1321 ccagccctcc gggacgccag cgcgaataag agtgtcaccg gtggtattac gacgtaacca
     1381 gtcagttaaa agagcccgct tgtcagccgg aagagcatcg cccaacacga atgcgcgcaa
     1441 acttgttgct aatgcacgag gggtactggt gtcacgcggg tcgcctggga cggcttcatt
     1501 cagctctgtt tcgtaacgat cagcgcgagt tacgtcatcc ccgatctctc tcaatgcagc
     1561 ctcgaagcca gccggtcctc ctaactcacg cagaagtaag ttgccagcag tgttatcact
     1621 ataacggacg gcagcgtcgc ataattctct caatgtcatg ccggtgtcga catgcttttc
     1681 tgttattggt gaatgggtaa ccaagtcatc acgagtataa gtgacgacct cgtcgagctc
     1741 ctcaagcccg gcgctgttac gttgtaatac cgcaccagca gccagggctt taaaagtgct
     1801 tgcataagca aaacgttcat cagcgcgatg agcctcgacg gtacgacccg tgccagtatc
     1861 cagtgcatat acacctaatc gtgcgtcgaa ctcacgttct aaacgtgcga attccaccag
     1921 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     1981 acgaaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     2041 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     2101 tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac
     2161 attaacctat aaaaataggc gtatcacgag gcagaatttc agataaaaaa aatccttagc
     2221 tttcgctaag gatgatttct ggaattcgcg gccgctgcta gccgtaaagg agaagaactt
     2281 ttcactggag ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg gcacaaattt
     2341 tctgtcagtg gagagggtga aggtgatgca acatacggaa aacttaccct taaatttatt
     2401 tgcactactg gaaaactacc tgttccatgg ccaacacttg tcactacttt cggttatggt
     2461 gttcaatgct ttgcgagata cccagatcat atgaaacagc atgacttttt caagagtgcc
     2521 atgcccgaag gttatgtaca ggaaagaact atatttttca aagatgacgg gaactacaag
     2581 acacgtgctg aagtcaagtt tgaaggtgat acccttgtta atagaatcga gttaaaaggt
     2641 attgatttta aagaagatgg aaacattctt ggacacaaat tggaatacaa ctataactca
     2701 cacaatgtat acatcatggc agacaaacaa aagaatggaa tcaaagttaa cttcaaaatt
     2761 agacacaaca ttgaagatgg aagcgttcaa ctagcagacc attatcaaca aaatactcca
     2821 attggcgatg gccctgtcct tttaccagac aaccattacc tgtccacaca atctgccctt
     2881 tcgaaagatc ccaacgaaaa gagagaccac atggtccttc ttgagtttgt aacagctgct
     2941 gggattacac atggcatgga tgaactatac aaataataat actagagcca ggcatcaaat
     3001 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
     3061 cgctctctac tagagtcaca ctggctcacc ttcgggtggg cctttctgcg tttatatact
     3121 agtagcggcc gctgcagtcc ggcaaaaaag ggcaaggtgt caccaccctg ccctttttct
     3181 ttaaaaccga aaagattact tcgcgttatg caggcttcct cgctcactga ctcgctgcgc
     3241 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     3301 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     3361 aaccgtaaaa aggccgcgtt gctggcgttt ttccacaggc tccgcccccc tgacgagcat
     3421 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     3481 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     3541 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtag
//
