LOCUS       sequence-491529-        6373 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  GGA cloning his-mscarletI3-AGGA-promoter-ccdb-TTCC.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6373)
  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 6373)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 4, 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: cwby0012
FEATURES             Location/Qualifiers
     source          1..6373
                     /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)"
     CDS             380..397
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1211..1516
                     /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     1395..1414
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(1585..1603)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1599..1646
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1683..2138
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1770..1789)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1980..2001
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2231..3046)
                     /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(2231..3046)
                     /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     2954..2973
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3038..3057
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3168..3756
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3657..3676
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3910..3927
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3942..4084
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4010..4029)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4170..4192
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4186..4377)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5186..6268)
                     /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     6230..6249
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6269..6346)
                     /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 catatgagcc atcatcatca ccatcatggc atggatagca ccgaagcggt
      421 gattaaagaa tttatgcgct ttaaagtgca tatggaaggc agcatgaacg gccatgaatt
      481 tgaaattgaa ggtgaaggcg aaggccgccc gtatgaaggc acccagaccg cgaaactgaa
      541 agtgaccaaa ggcggcccgc tgccgtttag ctgggatatt ctgagcccgc agtttatgta
      601 tggcagccgc gcgtttatta aacatccggc ggatatcccg gattattgga aacagagctt
      661 tccggaaggt tttaaatggg aacgcgtgat gatctttgaa gatggtggca ccgtgagcgt
      721 gacccaggat accagcctgg aagatggcac cctgatttat aaagtgaaac tgcgcggcgg
      781 caactttccg ccggatggcc cggtgatgca gaaacgcacc atgggctggg aagcgagcac
      841 cgaacgcctg tatccggaag atgtggtgct gaaaggcgat attaaaatgg cgctgcgcct
      901 gaaagatggc ggccgctatc tggcggattt taaaaccacc tataaagcga agaaaccggt
      961 gcagatgccg ggcgccttta acattgatcg caaactggat attaccagcc ataacgaaga
     1021 ttataccgtg gtggaacagt atgaacgcag cgtggcgcgc catagcaccg gcggtggcag
     1081 cggcagcggt ggctcaggag gagaccgaaa gcgtattaca gtgacagttg acagcgacag
     1141 ctatcagttg ctcaaggcat atatgatgtc aatatctccg gtctggtatg cacaaccaag
     1201 aatgaagccc atgcagttta aggtttacac ctataaaaga gagagccgtt atcgtctgtt
     1261 tgtggatgta cagagtgata ttattgacac gcccgggcga cggatggtga tccccctggc
     1321 cagtgcacgt ctgctgtcag ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg
     1381 ggatgaaagc tggcgcatga tgaccaccga tatggccagt gtgccggttt ccgttatcgg
     1441 ggaagaagtg gctgatctca gccaccgcga aaatgacatc aaaaacgcca ttaacctgat
     1501 gttctgggga atatgaacgg tctcgttcct aatgagatcc ggctgctaac aaagcccgaa
     1561 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     1621 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
     1681 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     1741 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     1801 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     1861 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     1921 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     1981 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     2041 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     2101 taacgcgaat tttaacaaaa tattaacgtt tacaatttca ggtggcactt ttcggggaaa
     2161 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2221 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     2281 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     2341 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     2401 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     2461 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
     2521 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     2581 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa
     2641 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     2701 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     2761 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     2821 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     2881 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     2941 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     3001 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
     3061 tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt
     3121 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     3181 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     3241 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     3301 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     3361 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     3421 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     3481 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     3541 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     3601 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     3661 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     3721 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     3781 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     3841 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     3901 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     3961 cttacgcatc tgtgcggtat ttcacaccgc atatatggtg cactctcagt acaatctgct
     4021 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     4081 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     4141 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     4201 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     4261 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     4321 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     4381 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     4441 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     4501 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     4561 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     4621 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     4681 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     4741 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     4801 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc
     4861 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     4921 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     4981 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     5041 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     5101 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     5161 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     5221 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     5281 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     5341 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     5401 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     5461 ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     5521 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     5581 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     5641 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     5701 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     5761 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     5821 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     5881 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     5941 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     6001 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     6061 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     6121 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     6181 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     6241 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     6301 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     6361 tctcccttat gcg
//
