LOCUS       sequence-491748-        6285 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  GGA cloning sfGFP-AGGA-ccdb-TTCC-HIS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6285)
  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 6285)
  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: cwby0003
FEATURES             Location/Qualifiers
     source          1..6285
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     41..60
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        41..59
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    60..84
                     /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             99..121
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             129..842
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             864..1169
                     /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     1048..1067
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1183..1200
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1256..1274)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1270..1317
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1354..1782
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1441..1460)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1651..1672
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1901..2716)
                     /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(1901..2716)
                     /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     2624..2643
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2708..2727
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2838..3426
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3327..3346
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3580..3597
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3612..3751
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3677..3696)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3837..3859
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3853..4044)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4853..5935)
                     /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     5897..5916
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5936..6013)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6219..6238
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ccggcgtaga ggatcgagat cgatctcgat cccgcgaaat taatacgact cactataggg
       61 gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag
      121 atatacatat gagcaaaggt gaagaactgt ttaccggcgt tgttccgatc ctggttgaac
      181 tggacggtga cgtgaacggt cataaattct ccgtacgtgg tgaaggtgag ggtgacgcga
      241 ccaacggtaa gctgactctg aaattcatct gcaccaccgg caaactgccg gttccgtggc
      301 cgacgctggt tacgaccctg acctacggtg ttcagtgctt cgcgcgttac ccggaccata
      361 tgaagcagca cgacttcttc aaatctgcga tgccggaagg ttacgttcag gaacgtacca
      421 tctctttcaa agacgacggt acctacaaaa cccgtgcgga agttaaattc gaaggcgaca
      481 ccctggttaa tcgtatcgaa ctgaaaggta tcgacttcaa ggaagacggc aatattctgg
      541 gtcacaaact ggaatacaac ttcaactctc acaatgttta catcaccgcg gacaaacaga
      601 aaaatggtat caaagcaaat ttcaaaatcc gtcataacgt tgaggacggc tctgtacaac
      661 tggcggacca ctaccaacaa aacaccccga ttggtgacgg tccggtcctg ctgccggaca
      721 accattacct gtctacccag tctgttctgt ctaaagaccc gaacgaaaaa cgtgaccaca
      781 tggttctgct ggaattcgtt accgcagcgg gtatcaccca cggtatggac gagctgtata
      841 aaggcagctc aggaggagac cgaatgcagt ttaaggttta cacctataaa agagagagcc
      901 gttatcgtct gtttgtggat gtacagagtg atattattga cacgcccggg cgacggatgg
      961 tgatccccct ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg
     1021 tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc agtgtgccgg
     1081 tttccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg
     1141 ccattaacct gatgttctgg ggaatatgaa cggtctcgtt cccaccacca ccaccaccac
     1201 taatgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     1261 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     1321 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ccgtagcggc gcattaagcg
     1381 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     1441 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     1501 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     1561 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     1621 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     1681 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     1741 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     1801 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     1861 ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca
     1921 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
     1981 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
     2041 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
     2101 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
     2161 aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
     2221 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata
     2281 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
     2341 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
     2401 ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
     2461 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
     2521 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
     2581 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     2641 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc
     2701 gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg
     2761 ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     2821 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     2881 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     2941 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     3001 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     3061 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     3121 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     3181 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     3241 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     3301 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     3361 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     3421 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     3481 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     3541 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     3601 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     3661 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     3721 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     3781 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     3841 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     3901 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     3961 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     4021 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     4081 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     4141 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     4201 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     4261 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     4321 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     4381 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     4441 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     4501 gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     4561 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     4621 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     4681 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     4741 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     4801 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     4861 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4921 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     4981 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     5041 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     5101 tgtcttcggt atcgtcgtat cccactaccg agatgtccgc accaacgcgc agcccggact
     5161 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     5221 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     5281 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     5341 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     5401 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     5461 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     5521 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     5581 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     5641 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     5701 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     5761 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     5821 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     5881 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     5941 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     6001 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     6061 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     6121 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     6181 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     6241 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgt
//
