LOCUS       sequence-493926-        6390 bp    DNA     circular SYN 25-NOV-2025
DEFINITION  GGA cloning AGGA-promoter-ccdb-TTCC-GS-C6-79-10-SNAC-6xhis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6390)
  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 6390)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 25, 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: cwby0072
FEATURES             Location/Qualifiers
     source          1..6390
                     /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             1487..1501
                     /codon_start=1
                     /product="tag for protein cleavage using Ni2+ ion (Dang et 
                     al., 2019)"
                     /label=SNAC tag
                     /translation="GSHHW"
     CDS             1511..1528
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1584..1602)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1598..1645
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1682..2137
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1769..1788)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1979..2000
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2229..3044)
                     /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(2229..3044)
                     /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     2952..2971
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3036..3055
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3166..3754
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3655..3674
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3908..3925
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3940..4079
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4005..4024)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4165..4187
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4181..4372)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGXDGENL"
     CDS             complement(5181..6263)
                     /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     6225..6244
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6264..6341)
                     /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 ggtagctctg atgaagaaga agcccgcgaa ttagaagaac gcgcgcgcga
      841 agccgcgaaa cgtgcaattg aagccgctaa acgcacaggc gacccgcgcg tgcgcgaact
      901 tgcagaagaa ttagttaaac tggcgatttg ggcagcggtg gaagtgtggc tggatccgag
      961 cagcagcgat gttaacgaag cgctgaaatt gattgtggaa gcgattgaag ctgctgtgcg
     1021 tgcgttggaa gccgcagaac gtacgggtga tccggaagtt cgtgaattag cgcgtgaatt
     1081 ggtgcgcctg gcggttgaag cggcagaaga ggttcagcgc aacccgagta gtagcgatgt
     1141 gaatgaagcc ctgaaactga ttgtgattgc cattgaggcg gcggttcgcg ctttagaagc
     1201 cgccgaacgt accggtgatc cagaagtgcg cgagctggca cgtgaactgg tgcgtttagc
     1261 ggtagaagcg gcggaagaag tgcagcgtaa cccgagctcc gaagaagtga acgaggcgct
     1321 gcgcaaaatt attaaactga ttctgttcgc ggtgatggtg ctggaattgg ccgaagaaat
     1381 tggcgatccg acctggcgtg aaatggcgcg tcgtgcagtg cgtgaagcag tggaactggc
     1441 tgaagaagtc cagcgcgatc cgtccggttg gctgggccat ggatcaggca gccatcattg
     1501 gggcagcacc caccaccacc accaccacta atgagatccg gctgctaaca aagcccgaaa
     1561 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     1621 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg
     1681 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     1741 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     1801 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     1861 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     1921 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     1981 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     2041 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     2101 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     2161 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     2221 attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     2281 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     2341 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     2401 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     2461 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     2521 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     2581 cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca
     2641 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     2701 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     2761 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     2821 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     2881 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     2941 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     3001 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta
     3061 ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc
     3121 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     3181 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     3241 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     3301 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3361 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3421 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3481 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3541 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3601 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3661 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3721 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3781 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3841 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3901 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     3961 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     4021 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     4081 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     4141 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     4201 yaccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     4261 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     4321 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     4381 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     4441 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     4501 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     4561 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     4621 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     4681 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     4741 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     4801 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa
     4861 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     4921 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     4981 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     5041 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     5101 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     5161 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5221 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     5281 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     5341 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     5401 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     5461 atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     5521 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     5581 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     5641 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     5701 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     5761 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     5821 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     5881 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     5941 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     6001 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     6061 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     6121 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     6181 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     6241 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     6301 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     6361 cttatgcgac tcctgcatta ggaagcagcc
//
