LOCUS       sequence-492958-        5886 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GGA cloning AGGA-promoter-ccdb-TTCC-GS-SB175-SNAC-6xhis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5886)
  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 5886)
  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: cwby0041
FEATURES             Location/Qualifiers
     source          1..5886
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     179..198
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     286..305
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        286..304
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    305..329
                     /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             344..366
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             494..799
                     /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     678..697
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1005..1019
                     /codon_start=1
                     /product="tag for protein cleavage using Ni2+ ion (Dang et 
                     al., 2019)"
                     /label=SNAC tag
                     /translation="GSHHW"
     CDS             1029..1046
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1102..1120)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1116..1163
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1200..1655
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1287..1306)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1497..1518
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1747..2562)
                     /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(1747..2562)
                     /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     2470..2489
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2554..2573
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2684..3272
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3173..3192
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3426..3443
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3458..3597
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3523..3542)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3683..3705
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3699..3890)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4699..5781)
                     /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     5743..5762
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5782..5859)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
       61 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
      121 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
      181 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
      241 tgcgtccggc gtagaggatc gagatcgatc tcgatcccgc gaaattaata cgactcacta
      301 taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga
      361 aggagatata catatgtcag gaggagaccg aaagcgtatt acagtgacag ttgacagcga
      421 cagctatcag ttgctcaagg catatatgat gtcaatatct ccggtctggt atgcacaacc
      481 aagaatgaag cccatgcagt ttaaggttta cacctataaa agagagagcc gttatcgtct
      541 gtttgtggat gtacagagtg atattattga cacgcccggg cgacggatgg tgatccccct
      601 ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg tggtgcatat
      661 cggggatgaa agctggcgca tgatgaccac cgatatggcc agtgtgccgg tttccgttat
      721 cggggaagaa gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg ccattaacct
      781 gatgttctgg ggaatatgaa cggtctcgtt ccggtagcga agcgctggaa gaactggaga
      841 aagcgctgcg cgaactgaag aaaagcaccg acgaactgga acgcagcacc gaagaactgg
      901 aaaagaaccc gagcgaagat gcgctggttg aaaacaaccg cctgattgtg gaaaacaaca
      961 aaattattgt ggaagtgctg cgcattattg cgaaagtgct gaaaggatca catcattggg
     1021 gcagcaccca ccaccaccac caccactaat gagatccggc tgctaacaaa gcccgaaagg
     1081 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     1141 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga
     1201 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     1261 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     1321 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     1381 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     1441 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     1501 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     1561 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     1621 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
     1681 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat
     1741 taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     1801 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     1861 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     1921 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     1981 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac
     2041 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     2101 tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
     2161 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     2221 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
     2281 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     2341 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     2401 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     2461 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     2521 tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact
     2581 gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt
     2641 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2701 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     2761 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     2821 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     2881 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     2941 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3001 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3061 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3121 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3181 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     3241 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3301 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     3361 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     3421 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     3481 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     3541 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     3601 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     3661 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     3721 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     3781 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     3841 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     3901 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     3961 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     4021 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     4081 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     4141 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     4201 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     4261 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     4321 gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg
     4381 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     4441 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     4501 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     4561 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     4621 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     4681 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4741 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     4801 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     4861 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     4921 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     4981 gtccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     5041 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     5101 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     5161 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     5221 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     5281 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     5341 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     5401 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     5461 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     5521 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     5581 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     5641 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     5701 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     5761 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     5821 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     5881 tatgcg
//
