LOCUS       sequence-493183-        5997 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GGA cloning AGGA-H8-GS-promoter-ccdb-TTCC-SNAC-6xhis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5997)
  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 5997)
  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: cwby0083
FEATURES             Location/Qualifiers
     source          1..5997
                     /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             775..1080
                     /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     959..978
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1094..1108
                     /codon_start=1
                     /product="tag for protein cleavage using Ni2+ ion (Dang et 
                     al., 2019)"
                     /label=SNAC tag
                     /translation="GSHHW"
     CDS             1118..1135
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1191..1209)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1205..1252
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1289..1744
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1376..1395)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1586..1607
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1836..2651)
                     /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(1836..2651)
                     /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     2559..2578
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2643..2662
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2773..3361
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3262..3281
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3515..3532
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3547..3686
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3612..3631)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3772..3794
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3788..3979)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4788..5870)
                     /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     5832..5851
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5871..5948)
                     /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 tatgagcgcg
      361 gaagaactgc tgcgccgcag ccgcgaatat ctgaagaaag tgaaagaaga acaggagcgc
      421 aaagcgaaag agtttcagga actgctgaaa gaactgagcg aacgcagcga agaactgatt
      481 cgcgaactgg aagagaaagg cgcggcgagc gaagcggaac tggcgcgtat gaaacagcag
      541 catatgaccg cgtatttaga agcgcagctg accgcctggg aaattgaaag caaatcgaaa
      601 attgcgctgc tggaactgca gcagaaccag ctgaacctgg aactgcgcca tattggatca
      661 ggaggagacc gaaagcgtat tacagtgaca gttgacagcg acagctatca gttgctcaag
      721 gcatatatga tgtcaatatc tccggtctgg tatgcacaac caagaatgaa gcccatgcag
      781 tttaaggttt acacctataa aagagagagc cgttatcgtc tgtttgtgga tgtacagagt
      841 gatattattg acacgcccgg gcgacggatg gtgatccccc tggccagtgc acgtctgctg
      901 tcagataaag tctcccgtga actttacccg gtggtgcata tcggggatga aagctggcgc
      961 atgatgacca ccgatatggc cagtgtgccg gtttccgtta tcggggaaga agtggctgat
     1021 ctcagccacc gcgaaaatga catcaaaaac gccattaacc tgatgttctg gggaatatga
     1081 acggtctcgt tccggcagcc atcattgggg cagcacccac caccaccacc accactaatg
     1141 agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     1201 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     1261 aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     1321 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     1381 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     1441 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     1501 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     1561 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     1621 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     1681 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     1741 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     1801 tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa
     1861 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     1921 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     1981 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     2041 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt
     2101 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
     2161 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga
     2221 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     2281 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     2341 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
     2401 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     2461 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     2521 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     2581 tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga
     2641 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
     2701 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     2761 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     2821 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     2881 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     2941 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3001 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3061 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3121 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3181 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3241 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3301 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3361 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     3421 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     3481 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     3541 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     3601 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     3661 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     3721 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     3781 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     3841 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     3901 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     3961 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     4021 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     4081 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     4141 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     4201 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     4261 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     4321 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     4381 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     4441 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     4501 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     4561 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     4621 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     4681 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     4741 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     4801 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     4861 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
     4921 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
     4981 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
     5041 tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta
     5101 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
     5161 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
     5221 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
     5281 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
     5341 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
     5401 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
     5461 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
     5521 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
     5581 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
     5641 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
     5701 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
     5761 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
     5821 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc
     5881 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg
     5941 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcc
//
