LOCUS       sequence-493167-        5899 bp    DNA     circular SYN 18-NOV-2025
DEFINITION  GGA cloning AGGA-HALC2_065-GS-promoter-ccdb-TTCC-SNAC-6xhis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5899)
  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 5899)
  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: cwby0053
FEATURES             Location/Qualifiers
     source          1..5899
                     /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             699..1004
                     /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     883..902
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1036..1053
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1115..1133)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1129..1176
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1213..1668
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1300..1319)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1510..1531
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1760..2575)
                     /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(1760..2575)
                     /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     2483..2502
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2567..2586
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2697..3285
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3186..3205
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3439..3456
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3471..3610
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3536..3555)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3696..3718
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3712..3903)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4712..5794)
                     /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     5756..5775
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5795..5872)
                     /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 catatgagcg aagaagaaaa gccgatcgtt attgatctga acaaaaccat
      421 tgaacgcgat ggccgcaaag tgaaactggt gcgcgcgacc attaccgtgg atccggaaac
      481 caacaccatt accattgata ttgaatacga gggcggcccg attaccaaag aagatttgct
      541 ggaagcgttt aaactggcgg cgagcaaact gggatcagga ggagaccggt aataaaaagc
      601 gtattacagt gacagttgac agcgacagct atcagttgct caaggcatat atgatgtcaa
      661 tatctccggt ctggtatgca caaccaagaa tgaagcccat gcagtttaag gtttacacct
      721 ataaaagaga gagccgttat cgtctgtttg tggatgtaca gagtgatatt attgacacgc
      781 ccgggcgacg gatggtgatc cccctggcca gtgcacgtct gctgtcagat aaagtctccc
      841 gtgaacttta cccggtggtg catatcgggg atgaaagctg gcgcatgatg accaccgata
      901 tggccagtgt gccggtttcc gttatcgggg aagaagtggc tgatctcagc caccgcgaaa
      961 atgacatcaa aaacgccatt aacctgatgt tctggggaat atgaacggtc tcgttcccat
     1021 cattggggca gcacccacca ccaccaccac cactaatgat aatgagatcc ggctgctaac
     1081 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
     1141 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
     1201 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     1261 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     1321 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     1381 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     1441 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     1501 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     1561 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     1621 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     1681 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     1741 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
     1801 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     1861 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     1921 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     1981 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
     2041 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     2101 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
     2161 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     2221 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     2281 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     2341 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     2401 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     2461 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     2521 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     2581 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     2641 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     2701 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     2761 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     2821 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     2881 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     2941 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     3001 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     3061 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     3121 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     3181 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     3241 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     3301 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     3361 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     3421 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     3481 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     3541 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     3601 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     3661 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     3721 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     3781 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     3841 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     3901 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     3961 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     4021 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     4081 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     4141 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     4201 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     4261 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     4321 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat
     4381 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc
     4441 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct
     4501 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag
     4561 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     4621 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     4681 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     4741 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     4801 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     4861 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     4921 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     4981 ccactaccga gatgtccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     5041 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
     5101 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     5161 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     5221 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     5281 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
     5341 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     5401 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     5461 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     5521 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     5581 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     5641 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     5701 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     5761 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     5821 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     5881 cgacgctctc ccttatgcg
//
