LOCUS       sequence-493180-        6306 bp    DNA     circular SYN 18-NOV-2025
DEFINITION  GGA cloning AGGA-promoter-ccdb-TTCC-GS-C4-71-8-SNAC-6xhis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6306)
  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 6306)
  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: cwby0069
FEATURES             Location/Qualifiers
     source          1..6306
                     /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             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             1403..1417
                     /codon_start=1
                     /product="tag for protein cleavage using Ni2+ ion (Dang et 
                     al., 2019)"
                     /label=SNAC tag
                     /translation="GSHHW"
     CDS             1427..1444
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1500..1518)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1514..1561
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1598..2053
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1685..1704)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1895..1916
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2145..2960)
                     /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(2145..2960)
                     /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     2868..2887
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2952..2971
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3082..3670
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3571..3590
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3824..3841
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3856..3995
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3921..3940)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4081..4103
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4097..4288)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5097..6179)
                     /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     6141..6160
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6180..6257)
                     /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 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 ggtccggaag aaattctgga acgcgcccgc gaaagcctgg aacgtgcgcg
      841 cgaagcgagc gaacgcggcg atgaagaaga atttcgcaaa gcggcggaaa aggcgctgga
      901 actggccaaa cgtctggtgg aacaggcgaa gaaagaaggg gatccgtgga tggtgatgtg
      961 ggcggccctg gtggccctgt gggtggcgct gctggcgctg cgcaacggtg ataaagaggt
     1021 gttcaagaaa gcggccgaaa gcgccctgga agtggcgaaa cgcttagtgg aagtggcgtc
     1081 aaaagaaggt gatccggaaa tggtgctgct ggcggcgtgg gttgcgctgt ttgtggcgtg
     1141 gctggcgtgg ctgtttggcg ataaagaagt gtttaagaaa gccgcggaaa gcgcgctgga
     1201 agttgcgaaa cgcctggttg aagtggccag caaagaaggc gatccggaac tggtggaaga
     1261 agcggcgaaa gtggcagaag aagttgagaa actggcggag aaacagggcg acgaagaagt
     1321 gcgcgagaaa gcgtgggaaa cctggatgga agtgtggctg ctgtggctgg aagtgcgtct
     1381 gcgtaaaggc ggcggcggat caggcagcca tcattggggc agcacccacc accaccacca
     1441 ccactaatga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
     1501 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     1561 gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta
     1621 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     1681 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     1741 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     1801 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     1861 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     1921 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     1981 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     2041 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     2101 ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg
     2161 agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa
     2221 agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc
     2281 tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg
     2341 tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat
     2401 ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca
     2461 tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga
     2521 aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg
     2581 aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg
     2641 aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata
     2701 aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca
     2761 tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg
     2821 ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat
     2881 ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt
     2941 tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt
     3001 attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3061 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3121 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3181 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     3241 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3301 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     3361 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     3421 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     3481 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     3541 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     3601 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     3661 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     3721 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     3781 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     3841 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3901 ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     3961 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     4021 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     4081 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     4141 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
     4201 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     4261 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     4321 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     4381 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     4441 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     4501 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     4561 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     4621 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     4681 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     4741 tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
     4801 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
     4861 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
     4921 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     4981 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     5041 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     5101 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     5161 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     5221 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     5281 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     5341 gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg
     5401 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     5461 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     5521 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     5581 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     5641 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     5701 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     5761 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     5821 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     5881 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     5941 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     6001 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     6061 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     6121 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     6181 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     6241 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     6301 gcagcc
//
