LOCUS       sequence-489558-        6418 bp    DNA     circular SYN 16-OCT-2025
DEFINITION  GGA cloning his-muGFP-AGGA-promoter-ccdb-TTCC.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6418)
  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 6418)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 16, 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: cwby0013
FEATURES             Location/Qualifiers
     source          1..6418
                     /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             1256..1561
                     /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     1440..1459
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(1630..1648)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1644..1691
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1728..2183
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1815..1834)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2025..2046
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2276..3091)
                     /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(2276..3091)
                     /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     2999..3018
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3083..3102
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3213..3801
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3702..3721
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3955..3972
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3987..4129
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4055..4074)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4215..4237
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4231..4422)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5231..6313)
                     /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     6275..6294
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6314..6391)
                     /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 atgagcagca aaggcgaaga
      421 actgtttacc ggcgtggtgc cgattctggt ggaactggat ggcgatgtga acggccataa
      481 atttagcgtg cgcggcgaag gcgaaggtga tgcgaccaac ggtaaactga ccctgaaatt
      541 tatttgcacc accggcaaac tgccggtgcc gtggccgacc ctggttacta ccttaaccta
      601 tggcgttctg tgctttagcc gctatccgga tcatatgaaa cgccatgatt tctttaaaag
      661 cgcgatgccg gaaggctatg tgcaggaacg caccattagc tttaaagatg atggcaccta
      721 taaaacccgc gccgaagtga aatttgaagg cgataccctg gtgaaccgca ttgaactgaa
      781 aggcatcgat tttaaagaag acggcaacat cctgggtcat aaactggaat ataacttcaa
      841 cagccataac gtgtatatta ccgcggataa acagaagaac ggcattaagg cgtatttcaa
      901 aattcgccac aacgttgaag atggcagcgt gcagctggcg gatcactatc agcagaacac
      961 cccgatcggc gatggcccag tgctgctgcc ggataaccat tatctgagca cccagagcgt
     1021 gctgagcaaa gatccgaacg aaaagcgtga tcatatggtg ctgctggaag atgtgaccgc
     1081 ggcgggcatt acccatggca tggatgaact gtataaaggt ggcagcggca gcggtggctc
     1141 aggaggagac cgaaagcgta ttacagtgac agttgacagc gacagctatc agttgctcaa
     1201 ggcatatatg atgtcaatat ctccggtctg gtatgcacaa ccaagaatga agcccatgca
     1261 gtttaaggtt tacacctata aaagagagag ccgttatcgt ctgtttgtgg atgtacagag
     1321 tgatattatt gacacgcccg ggcgacggat ggtgatcccc ctggccagtg cacgtctgct
     1381 gtcagataaa gtctcccgtg aactttaccc ggtggtgcat atcggggatg aaagctggcg
     1441 catgatgacc accgatatgg ccagtgtgcc ggtttccgtt atcggggaag aagtggctga
     1501 tctcagccac cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct ggggaatatg
     1561 aacggtctcg ttcctaatga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     1621 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     1681 ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag
     1741 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     1801 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     1861 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     1921 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     1981 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     2041 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     2101 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     2161 caaaatatta acgtttacaa tttcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     2221 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga
     2281 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     2341 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
     2401 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
     2461 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
     2521 ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat
     2581 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
     2641 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     2701 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt
     2761 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag
     2821 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     2881 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     2941 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat
     3001 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag
     3061 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag
     3121 cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     3181 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     3241 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     3301 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     3361 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     3421 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     3481 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     3541 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     3601 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     3661 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     3721 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     3781 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     3841 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     3901 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     3961 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     4021 ggtatttcac accgcatata tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     4081 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     4141 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     4201 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     4261 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     4321 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     4381 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     4441 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     4501 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     4561 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     4621 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     4681 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     4741 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     4801 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     4861 cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt
     4921 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
     4981 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc
     5041 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
     5101 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     5161 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     5221 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     5281 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     5341 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     5401 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     5461 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atgtccgcac
     5521 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     5581 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     5641 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     5701 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     5761 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     5821 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     5881 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     5941 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     6001 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     6061 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     6121 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     6181 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     6241 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     6301 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     6361 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcg
//
