LOCUS       sequence-493179-        6402 bp    DNA     circular SYN 18-NOV-2025
DEFINITION  GGA cloning AGGA-promoter-ccdb-TTCC-GS-C4-81-4-SNAC-6xhis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6402)
  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 6402)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 18, 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: cwby0070
FEATURES             Location/Qualifiers
     source          1..6402
                     /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"
     RBS             322..344
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             472..777
                     /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     656..675
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1499..1513
                     /codon_start=1
                     /product="tag for protein cleavage using Ni2+ ion (Dang et 
                     al., 2019)"
                     /label=SNAC tag
                     /translation="GSHHW"
     CDS             1523..1540
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1596..1614)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1610..1657
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1694..2149
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1781..1800)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1991..2012
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2241..3056)
                     /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(2241..3056)
                     /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     2964..2983
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3048..3067
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3178..3766
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3667..3686
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3920..3937
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3952..4091
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4017..4036)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4177..4199
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4193..4384)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5193..6275)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6237..6256
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6276..6353)
                     /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 cgattcccct ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgtcagga
      361 ggagaccgaa agcgtattac agtgacagtt gacagcgaca gctatcagtt gctcaaggca
      421 tatatgatgt caatatctcc ggtctggtat gcacaaccaa gaatgaagcc catgcagttt
      481 aaggtttaca cctataaaag agagagccgt tatcgtctgt ttgtggatgt acagagtgat
      541 attattgaca cgcccgggcg acggatggtg atccccctgg ccagtgcacg tctgctgtca
      601 gataaagtct cccgtgaact ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg
      661 atgaccaccg atatggccag tgtgccggtt tccgttatcg gggaagaagt ggctgatctc
      721 agccaccgcg aaaatgacat caaaaacgcc attaacctga tgttctgggg aatatgaacg
      781 gtctcgttcc ggtggcgaac tggaacgtga aagccgcgaa gcggaaaagc gtctgaaaga
      841 agcgcgcctg tttgcgtggg cggcgcgtct gctgggcgat ctgaaactgc tggcgaaagc
      901 gctgattgaa gaagcgcgtg cggtgcagga actggcgcgc gtggcgtgcg aacgcggcaa
      961 ccgcgatgaa gcgtgggatg cgtttgagaa agcgctggaa gtgttcgaag aagcggtgaa
     1021 agtgtcggaa gaagcacgcg aacaaggcga tgatgaagtg ctggcgttag cactgattgc
     1081 gattgcgctg gcggtgctgg cactggccga agtggcgtgc tgcctgggca ttagcgagct
     1141 ggcggaactg gcgtggaaaa tggcggaatg ggtgctggaa gaagcgcgca aagtgagcga
     1201 agaggcgcgt gaacagggcg acgatgaagt tctggcgctg gccctgattg ccattgccct
     1261 ggcggtcctg gccttagcgg aagtggcctg ctgccgtggc aacaaagaag aagccgaacg
     1321 tgcgtatgaa gatgcgcgcc gcgtggaaga ggaagcgcgt aaagtgaaag aaagcgcgga
     1381 agaacagggt gatagcgaag tgaaacgcct ggcagaagaa gcggaacagc tggcccgtga
     1441 agcgcgtcgc catgttcagg aatgccgcgg cggctggctg gaacatggca gcggatcagg
     1501 cagccatcat tggggcagca cccaccacca ccaccaccac taatgagatc cggctgctaa
     1561 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     1621 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     1681 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     1741 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     1801 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     1861 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     1921 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     1981 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     2041 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     2101 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     2161 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     2221 atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
     2281 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
     2341 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
     2401 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
     2461 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
     2521 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
     2581 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
     2641 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     2701 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     2761 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     2821 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     2881 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     2941 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     3001 atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca
     3061 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta
     3121 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     3181 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     3241 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     3301 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     3361 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     3421 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     3481 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     3541 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     3601 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     3661 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     3721 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     3781 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     3841 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     3901 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     3961 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca
     4021 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     4081 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     4141 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     4201 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
     4261 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
     4321 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
     4381 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
     4441 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
     4501 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
     4561 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
     4621 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg
     4681 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
     4741 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
     4801 gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca
     4861 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     4921 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     4981 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     5041 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     5101 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
     5161 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5221 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5281 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     5341 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     5401 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     5461 cccactaccg agatgtccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     5521 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     5581 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     5641 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     5701 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     5761 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     5821 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     5881 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     5941 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     6001 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     6061 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     6121 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     6181 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     6241 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     6301 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
     6361 tcgacgctct cccttatgcg actcctgcat taggaagcag cc
//
