LOCUS       sequence-493930-        5207 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GGA cloning AGGA-H6-GS-promoter-ccdb-TTCC-SNAC-6xhis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5207)
  AUTHORS   Qian J, Milles LF, Wicky BIM, Motmaen A, Li X, Kibler RD, Stewart L,
            Baker D
  TITLE     Accelerating protein design by scaling experimental characterization
  JOURNAL   bioRxiv 2025.08.05.668824; doi: 
            https://doi.org/10.1101/2025.08.05.668824
REFERENCE   2  (bases 1 to 5207)
  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: "bioRxiv 
            2025.08.05.668824; doi: https://doi.org/10.1101/2025.08.05.668824"
COMMENT     Sequence Label: cwby0082
FEATURES             Location/Qualifiers
     source          1..5207
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     157..176
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     264..283
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        264..282
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    283..307
                     /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             322..344
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             691..996
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     875..894
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1010..1024
                     /codon_start=1
                     /product="tag for protein cleavage using Ni2+ ion (Dang et 
                     al., 2019)"
                     /label=SNAC tag
                     /translation="GSHHW"
     CDS             1034..1051
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1107..1125)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1121..1168
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1205..1660
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1292..1311)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1502..1523
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1752..2567)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1752..2567)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2475..2494
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2559..2578
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2689..3277
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3178..3197
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3431..3448
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3463..3602
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3528..3547)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3688..3710
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3704..3895)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5042..5061
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5081..5158)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat
       61 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct
      121 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc
      181 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga
      241 gatcgatctc gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa
      301 caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgaccgaa
      361 gatgaaattc gcaaactgcg caagctgctg gaagaagcgg aaaagaaact gaagaaactg
      421 gaagataaaa cccgccgcag cgaagaaatt agcaaaaccg atgatgatcc gaaagcgcag
      481 agcctgcagc tgattgctga aagcctgatg ctgatcgcgg aaagtctgct gattattgcg
      541 attagcctgc tgctgagcag ccgcaacggc ggatcaggag gagaccgaaa gcgtattaca
      601 gtgacagttg acagcgacag ctatcagttg ctcaaggcat atatgatgtc aatatctccg
      661 gtctggtatg cacaaccaag aatgaagccc atgcagttta aggtttacac ctataaaaga
      721 gagagccgtt atcgtctgtt tgtggatgta cagagtgata ttattgacac gcccgggcga
      781 cggatggtga tccccctggc cagtgcacgt ctgctgtcag ataaagtctc ccgtgaactt
      841 tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga tatggccagt
      901 gtgccggttt ccgttatcgg ggaagaagtg gctgatctca gccaccgcga aaatgacatc
      961 aaaaacgcca ttaacctgat gttctgggga atatgaacgg tctcgttccg gcagccatca
     1021 ttggggcagc acccaccacc accaccacca ctaatgagat ccggctgcta acaaagcccg
     1081 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     1141 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa
     1201 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     1261 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     1321 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     1381 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     1441 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     1501 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     1561 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     1621 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     1681 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     1741 tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
     1801 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
     1861 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
     1921 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
     1981 tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt
     2041 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
     2101 attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa
     2161 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     2221 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
     2281 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     2341 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     2401 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
     2461 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     2521 tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta
     2581 ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt
     2641 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     2701 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     2761 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     2821 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     2881 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     2941 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     3001 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     3061 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     3121 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     3181 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     3241 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     3301 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     3361 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     3421 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     3481 ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct
     3541 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     3601 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     3661 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     3721 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     3781 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     3841 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     3901 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     3961 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     4021 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     4081 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     4141 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     4201 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     4261 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     4321 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga
     4381 taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga
     4441 gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa
     4501 agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga
     4561 taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc
     4621 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     4681 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4741 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt ggcaccacca
     4801 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     4861 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     4921 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     4981 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     5041 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     5101 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg
     5161 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcc
//
