LOCUS       sequence-491525-        6403 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  GGA cloning his-mBaoJin-AGGA-promoter-ccdb-TTCC.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6403)
  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 6403)
  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: cwby0014
FEATURES             Location/Qualifiers
     source          1..6403
                     /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             401..1102
                     /codon_start=1
                     /product="bright and photostable monomeric green 
                     fluorescent protein derived from StayGold (Zhang et al., 
                     2024)"
                     /label=mBaoJin
                     /label=mStayGold
                     /note="/vntifkey=21"
                     /translation="MVSKGEEENMASTPFKFQLKGTINGKSFTVEGEGEGNSHEGSHKG
                     KYVCTSGKLPMSWAALGTTFGYGMKYYTKYPSGLKNWFREVMPGGFTYDRHIQYKGDGS
                     IHAKHQHFMKNGTYHNIVEFTGQDFKENSPVLTGDMNVSLPNEVPQIPRDDGVECPVTL
                     LYPLLSDKSKYVEAHQYTICKPLHNQPAPDVPYHWIRKQYTQSKDDAEERDHICQSETL
                     EAHLKGMDELYK"
     CDS             1241..1546
                     /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     1425..1444
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(1615..1633)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1629..1676
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1713..2168
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1800..1819)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2010..2031
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2261..3076)
                     /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(2261..3076)
                     /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     2984..3003
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3068..3087
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3198..3786
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3687..3706
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3940..3957
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3972..4114
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4040..4059)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4200..4222
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4216..4407)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5216..6298)
                     /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     6260..6279
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6299..6376)
                     /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 atggtgagca aaggcgaaga
      421 agaaaatatg gcgagcaccc cgtttaaatt tcagctgaaa ggcaccatta acggcaaaag
      481 ctttaccgtg gaaggtgaag gcgaaggcaa cagccatgaa ggcagccata aaggcaaata
      541 tgtgtgcacc agcggcaaac tgccgatgag ctgggcggcg ctgggcacca cctttggcta
      601 tggcatgaaa tattatacca aatatccgag cggcctgaag aactggtttc gcgaagtgat
      661 gccgggcggc tttacctatg atcgccatat tcagtataaa ggtgatggca gcattcatgc
      721 gaaacatcag cattttatga agaatggcac ctatcataac attgtggaat ttaccggcca
      781 ggattttaaa gaaaacagcc cggtgctgac cggcgatatg aacgtgagcc tgccgaacga
      841 agtgccgcag attccgcgcg atgatggcgt ggaatgcccg gtgaccctgc tgtatccgct
      901 gctgagcgat aaaagcaaat acgtggaagc ccatcagtat accatttgca aaccgctgca
      961 taaccagccg gcgccggatg tgccgtatca ttggattcgc aaacagtaca cccagagcaa
     1021 agatgatgcg gaagaacgcg atcatatttg ccagagcgaa accctggaag cgcatctgaa
     1081 aggtatggat gaactgtata aaggtggcag cggcagcggt ggctcaggag gagaccgaaa
     1141 gcgtattaca gtgacagttg acagcgacag ctatcagttg ctcaaggcat atatgatgtc
     1201 aatatctccg gtctggtatg cacaaccaag aatgaagccc atgcagttta aggtttacac
     1261 ctataaaaga gagagccgtt atcgtctgtt tgtggatgta cagagtgata ttattgacac
     1321 gcccgggcga cggatggtga tccccctggc cagtgcacgt ctgctgtcag ataaagtctc
     1381 ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga
     1441 tatggccagt gtgccggttt ccgttatcgg ggaagaagtg gctgatctca gccaccgcga
     1501 aaatgacatc aaaaacgcca ttaacctgat gttctgggga atatgaacgg tctcgttcct
     1561 aatgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     1621 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     1681 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     1741 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     1801 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     1861 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     1921 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     1981 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     2041 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     2101 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt
     2161 tacaatttca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     2221 ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca
     2281 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
     2341 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
     2401 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
     2461 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
     2521 aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
     2581 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata
     2641 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
     2701 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
     2761 ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
     2821 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
     2881 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
     2941 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     3001 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc
     3061 gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg
     3121 ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     3181 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     3241 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     3301 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     3361 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     3421 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     3481 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     3541 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     3601 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     3661 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     3721 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     3781 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     3841 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     3901 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     3961 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     4021 atatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     4081 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     4141 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     4201 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     4261 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     4321 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     4381 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     4441 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     4501 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     4561 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     4621 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     4681 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     4741 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     4801 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     4861 catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt
     4921 ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag
     4981 cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag
     5041 cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac
     5101 gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     5161 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     5221 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5281 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     5341 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     5401 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     5461 agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg
     5521 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     5581 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     5641 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     5701 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     5761 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     5821 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     5881 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     5941 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     6001 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     6061 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     6121 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     6181 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     6241 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     6301 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
     6361 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcg
//
