LOCUS       sequence-491530-        5268 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GGA cloning cwby template vector pet29-ATG-PaQCI-TAAT_GA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5268)
  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 5268)
  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: cwby0004
FEATURES             Location/Qualifiers
     source          1..5268
                     /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)"
     primer_bind     complement(480..498)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      494..541
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      578..1033
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(665..684)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     875..896
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1126..1941)
                     /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(1126..1941)
                     /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     1849..1868
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1933..1952
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2063..2651
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2552..2571
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2805..2822
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2837..2979
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2905..2924)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3065..3087
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3081..3272)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4081..5163)
                     /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     5125..5144
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5164..5241)
                     /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 gcaggtggag gaaagcgaaa tggggcagca ccccacctgc
      421 ccactaatga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
      481 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
      541 gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta
      601 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
      661 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
      721 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
      781 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
      841 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
      901 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
      961 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     1021 acgtttacaa tttcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     1081 tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc
     1141 gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
     1201 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
     1261 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc
     1321 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
     1381 tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     1441 atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg
     1501 aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag
     1561 gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg
     1621 gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     1681 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc
     1741 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
     1801 gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     1861 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt
     1921 ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt
     1981 tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     2041 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     2101 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     2161 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     2221 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     2281 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     2341 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     2401 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     2461 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     2521 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     2581 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     2641 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     2701 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     2761 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     2821 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     2881 accgcatata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     2941 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
     3001 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     3061 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     3121 ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc
     3181 gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc
     3241 atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg
     3301 ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat
     3361 gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg
     3421 cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt
     3481 gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc
     3541 gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt
     3601 gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat
     3661 tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc
     3721 acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa
     3781 cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc
     3841 gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc
     3901 cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg
     3961 gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     4021 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     4081 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     4141 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     4201 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     4261 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     4321 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atgtccgcac caacgcgcag
     4381 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     4441 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     4501 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     4561 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     4621 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
     4681 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     4741 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     4801 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     4861 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     4921 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
     4981 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     5041 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     5101 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     5161 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     5221 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcg
//
