LOCUS       sequence-493132-        6300 bp    DNA     circular SYN 18-NOV-2025
DEFINITION  GGA cloning AGGA-C4-71-8-GS-promoter-ccdb-TTCC-SNAC-6xhis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6300)
  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 6300)
  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: cwby0077
FEATURES             Location/Qualifiers
     source          1..6300
                     /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             1078..1383
                     /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     1262..1281
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1397..1411
                     /codon_start=1
                     /product="tag for protein cleavage using Ni2+ ion (Dang et 
                     al., 2019)"
                     /label=SNAC tag
                     /translation="GSHHW"
     CDS             1421..1438
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1494..1512)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1508..1555
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1592..2047
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1679..1698)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1889..1910
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2139..2954)
                     /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(2139..2954)
                     /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     2862..2881
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2946..2965
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3076..3664
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3565..3584
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3818..3835
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3850..3989
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3915..3934)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4075..4097
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4091..4282)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5091..6173)
                     /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     6135..6154
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6174..6251)
                     /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 tatgccggaa
      361 gaaattctgg aacgcgcccg cgaaagcctg gaacgtgcgc gcgaagcgag cgaacgcggc
      421 gatgaagaag aatttcgcaa agcggcggaa aaggcgctgg aactggccaa acgtctggtg
      481 gaacaggcga agaaagaagg ggatccgtgg atggtgatgt gggcggccct ggtggccctg
      541 tgggtggcgc tgctggcgct gcgcaacggt gataaagagg tgttcaagaa agcggccgaa
      601 agcgccctgg aagtggcgaa acgcttagtg gaagtggcgt caaaagaagg tgatccggaa
      661 atggtgctgc tggcggcgtg ggttgcgctg tttgtggcgt ggctggcgtg gctgtttggc
      721 gataaagaag tgtttaagaa agccgcggaa agcgcgctgg aagttgcgaa acgcctggtt
      781 gaagtggcca gcaaagaagg cgatccggaa ctggtggaag aagcggcgaa agtggcagaa
      841 gaagttgaga aactggcgga gaaacagggc gacgaagaag tgcgcgagaa agcgtgggaa
      901 acctggatgg aagtgtggct gctgtggctg gaagtgcgtc tgcgtaaagg cggcggcgga
      961 tcaggaggag accgaaagcg tattacagtg acagttgaca gcgacagcta tcagttgctc
     1021 aaggcatata tgatgtcaat atctccggtc tggtatgcac aaccaagaat gaagcccatg
     1081 cagtttaagg tttacaccta taaaagagag agccgttatc gtctgtttgt ggatgtacag
     1141 agtgatatta ttgacacgcc cgggcgacgg atggtgatcc ccctggccag tgcacgtctg
     1201 ctgtcagata aagtctcccg tgaactttac ccggtggtgc atatcgggga tgaaagctgg
     1261 cgcatgatga ccaccgatat ggccagtgtg ccggtttccg ttatcgggga agaagtggct
     1321 gatctcagcc accgcgaaaa tgacatcaaa aacgccatta acctgatgtt ctggggaata
     1381 tgaacggtct cgttccggca gccatcattg gggcagcacc caccaccacc accaccacta
     1441 atgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
     1501 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     1561 aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     1621 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     1681 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     1741 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     1801 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     1861 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     1921 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     1981 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     2041 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     2101 aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc
     2161 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
     2221 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
     2281 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
     2341 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
     2401 agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
     2461 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg
     2521 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
     2581 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
     2641 gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
     2701 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
     2761 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
     2821 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
     2881 ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt
     2941 tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt
     3001 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     3061 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     3121 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     3181 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     3241 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     3301 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     3361 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     3421 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     3481 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     3541 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     3601 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     3661 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     3721 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     3781 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     3841 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa
     3901 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     3961 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     4021 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     4081 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     4141 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     4201 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     4261 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
     4321 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
     4381 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga
     4441 gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg
     4501 gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc
     4561 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg
     4621 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct
     4681 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc
     4741 gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg
     4801 cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca
     4861 ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg
     4921 ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag
     4981 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     5041 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     5101 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     5161 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
     5221 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
     5281 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
     5341 tcttcggtat cgtcgtatcc cactaccgag atgtccgcac caacgcgcag cccggactcg
     5401 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
     5461 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
     5521 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
     5581 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
     5641 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
     5701 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
     5761 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
     5821 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
     5881 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
     5941 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
     6001 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
     6061 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
     6121 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     6181 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     6241 tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc
//
