LOCUS       sequence-492962-        5868 bp    DNA     circular SYN 18-NOV-2025
DEFINITION  GGA cloning AGGA-C2-02802-GS-promoter-ccdb-TTCC-SNAC-6xhis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5868)
  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 5868)
  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: cwby0076
FEATURES             Location/Qualifiers
     source          1..5868
                     /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             646..951
                     /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     830..849
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             965..979
                     /codon_start=1
                     /product="tag for protein cleavage using Ni2+ ion (Dang et 
                     al., 2019)"
                     /label=SNAC tag
                     /translation="GSHHW"
     CDS             989..1006
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1062..1080)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1076..1123
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1160..1615
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1247..1266)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1457..1478
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1707..2522)
                     /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(1707..2522)
                     /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     2430..2449
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2514..2533
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2644..3232
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3133..3152
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3386..3403
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3418..3557
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3483..3502)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3643..3665
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3659..3850)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4659..5741)
                     /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     5703..5722
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5742..5819)
                     /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 tatggatctg
      361 gaagagcgct tcgaacgcct gtatgaaaag gcgaagaaac tggcggaaga acgcggcgac
      421 gaacgtgcgc gtcgcatgat tgaattactg cgccagctgt ttgaaaccgt gggcgatccg
      481 cgcattctgg aactgttaga actgctgctg cagctgctgg aaggcggatc aggaggagac
      541 cgaaagcgta ttacagtgac agttgacagc gacagctatc agttgctcaa ggcatatatg
      601 atgtcaatat ctccggtctg gtatgcacaa ccaagaatga agcccatgca gtttaaggtt
      661 tacacctata aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt
      721 gacacgcccg ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa
      781 gtctcccgtg aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc
      841 accgatatgg ccagtgtgcc ggtttccgtt atcggggaag aagtggctga tctcagccac
      901 cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct ggggaatatg aacggtctcg
      961 ttccggcagc catcattggg gcagcaccca ccaccaccac caccactaat gagatccggc
     1021 tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc
     1081 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
     1141 atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     1201 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     1261 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     1321 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     1381 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     1441 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     1501 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     1561 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
     1621 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     1681 atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc
     1741 aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa
     1801 ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt
     1861 ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca
     1921 agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt
     1981 tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca
     2041 accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta
     2101 aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca
     2161 acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg
     2221 atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga
     2281 agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca
     2341 acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga
     2401 tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca
     2461 gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc
     2521 ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat
     2581 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     2641 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     2701 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     2761 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     2821 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     2881 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     2941 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     3001 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     3061 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     3121 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     3181 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     3241 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     3301 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     3361 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     3421 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc
     3481 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
     3541 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     3601 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     3661 agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt
     3721 ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct
     3781 ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct
     3841 gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga
     3901 tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg
     3961 aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc
     4021 agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc
     4081 atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac
     4141 tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc
     4201 agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc
     4261 aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg
     4321 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     4381 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     4441 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     4501 ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg
     4561 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     4621 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4681 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4741 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     4801 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     4861 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     4921 tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt aatggcgcgc
     4981 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     5041 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     5101 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     5161 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     5221 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     5281 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     5341 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     5401 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     5461 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     5521 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     5581 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     5641 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     5701 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     5761 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     5821 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagcc
//
