LOCUS       sequence-493131-        6396 bp    DNA     circular SYN 18-NOV-2025
DEFINITION  GGA cloning AGGA-C4-81-4-GS-promoter-ccdb-TTCC-SNAC-6xhis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6396)
  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 6396)
  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: cwby0078
FEATURES             Location/Qualifiers
     source          1..6396
                     /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             1174..1479
                     /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     1358..1377
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1493..1507
                     /codon_start=1
                     /product="tag for protein cleavage using Ni2+ ion (Dang et 
                     al., 2019)"
                     /label=SNAC tag
                     /translation="GSHHW"
     CDS             1517..1534
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1590..1608)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1604..1651
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1688..2143
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1775..1794)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1985..2006
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2235..3050)
                     /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(2235..3050)
                     /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     2958..2977
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3042..3061
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3172..3760
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3661..3680
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3914..3931
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3946..4085
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4011..4030)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4171..4193
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4187..4378)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5187..6269)
                     /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     6231..6250
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6270..6347)
                     /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 tatgggcgaa
      361 ctggaacgtg aaagccgcga agcggaaaag cgtctgaaag aagcgcgcct gtttgcgtgg
      421 gcggcgcgtc tgctgggcga tctgaaactg ctggcgaaag cgctgattga agaagcgcgt
      481 gcggtgcagg aactggcgcg cgtggcgtgc gaacgcggca accgcgatga agcgtgggat
      541 gcgtttgaga aagcgctgga agtgttcgaa gaagcggtga aagtgtcgga agaagcacgc
      601 gaacaaggcg atgatgaagt gctggcgtta gcactgattg cgattgcgct ggcggtgctg
      661 gcactggccg aagtggcgtg ctgcctgggc attagcgagc tggcggaact ggcgtggaaa
      721 atggcggaat gggtgctgga agaagcgcgc aaagtgagcg aagaggcgcg tgaacagggc
      781 gacgatgaag ttctggcgct ggccctgatt gccattgccc tggcggtcct ggccttagcg
      841 gaagtggcct gctgccgtgg caacaaagaa gaagccgaac gtgcgtatga agatgcgcgc
      901 cgcgtggaag aggaagcgcg taaagtgaaa gaaagcgcgg aagaacaggg tgatagcgaa
      961 gtgaaacgcc tggcagaaga agcggaacag ctggcccgtg aagcgcgtcg ccatgttcag
     1021 gaatgccgcg gcggctggct ggaacatggc agcggatcag gaggagaccg aaagcgtatt
     1081 acagtgacag ttgacagcga cagctatcag ttgctcaagg catatatgat gtcaatatct
     1141 ccggtctggt atgcacaacc aagaatgaag cccatgcagt ttaaggttta cacctataaa
     1201 agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga cacgcccggg
     1261 cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa
     1321 ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc
     1381 agtgtgccgg tttccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac
     1441 atcaaaaacg ccattaacct gatgttctgg ggaatatgaa cggtctcgtt ccggcagcca
     1501 tcattggggc agcacccacc accaccacca ccactaatga gatccggctg ctaacaaagc
     1561 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
     1621 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc
     1681 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     1741 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     1801 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     1861 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     1921 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     1981 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     2041 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     2101 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     2161 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     2221 tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata
     2281 tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca
     2341 ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca
     2401 acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca
     2461 ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact
     2521 tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta
     2581 ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta
     2641 caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca
     2701 cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg
     2761 agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat
     2821 tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg
     2881 ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca
     2941 cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg
     3001 gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt
     3061 gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga
     3121 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     3181 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     3241 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     3301 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     3361 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     3421 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     3481 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     3541 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     3601 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     3661 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     3721 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     3781 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     3841 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     3901 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     3961 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
     4021 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     4081 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     4141 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     4201 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     4261 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     4321 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     4381 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     4441 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     4501 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     4561 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     4621 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     4681 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     4741 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     4801 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg
     4861 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     4921 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     4981 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     5041 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     5101 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     5161 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     5221 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     5281 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     5341 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     5401 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     5461 accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     5521 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     5581 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     5641 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     5701 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     5761 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     5821 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     5881 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     5941 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     6001 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     6061 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     6121 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     6181 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     6241 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     6301 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     6361 ctctccctta tgcgactcct gcattaggaa gcagcc
//
