LOCUS       sequence-491749-        6408 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  GGA cloning his-TagGFP2-AGGA-promoter-ccdb-TTCC.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6408)
  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 6408)
  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: cwby0010
FEATURES             Location/Qualifiers
     source          1..6408
                     /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             1250..1555
                     /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     1434..1453
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(1624..1642)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1638..1685
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1722..2177
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1809..1828)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2019..2040
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2269..3084)
                     /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(2269..3084)
                     /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     2992..3011
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3076..3095
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3206..3794
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3695..3714
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3948..3965
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3980..4119
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4045..4064)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4205..4227
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4221..4412)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5221..6303)
                     /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     6265..6284
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6304..6381)
                     /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 atcatcacca tcatcatggc agcggcggcg aagaactgtt
      421 tgcgggcatt gttccggttc tgattgaact ggatggcgat gtgcatggcc ataaatttag
      481 cgtgcgcggt gaaggcgaag gcgatgcgga ttatggcaaa ctggaaatca aatttatttg
      541 caccacaggc aaactgccgg ttccgtggcc gaccctggtt accactctgt gctatggtat
      601 tcagtgcttt gcgcgctatc cggaacatat gaagatgaac gatttcttta aaagcgcgat
      661 gccggaaggc tatattcagg aacgcaccat tcagtttcag gatgatggca aatataaaac
      721 ccgtggcgaa gtgaaatttg aaggtgatac cctggtgaac cgcattgaac tgaaaggcaa
      781 agattttaaa gaagacggca acattctggg tcataaactg gaatacagct ttaacagcca
      841 taacgtgtat attcgcccgg ataaagcgaa caacggcctg gaagcgaact tcaaaacccg
      901 ccataacatt gaaggcggtg gcgtgcagct ggcggatcat tatcagacca acgtgccgtt
      961 aggcgatggc ccggtgctga ttccgattaa ccattatctg agcacccaga ccaaaattag
     1021 caaagatcgc aacgaagcgc gcgatcatat ggtgctgctg gaaagcttta gcgcgtgctg
     1081 ccatacccat ggcatggatg aactgtatcg tggcggcagc ggtagcggtg gctcaggagg
     1141 agaccgaaag cgtattacag tgacagttga cagcgacagc tatcagttgc tcaaggcata
     1201 tatgatgtca atatctccgg tctggtatgc acaaccaaga atgaagccca tgcagtttaa
     1261 ggtttacacc tataaaagag agagccgtta tcgtctgttt gtggatgtac agagtgatat
     1321 tattgacacg cccgggcgac ggatggtgat ccccctggcc agtgcacgtc tgctgtcaga
     1381 taaagtctcc cgtgaacttt acccggtggt gcatatcggg gatgaaagct ggcgcatgat
     1441 gaccaccgat atggccagtg tgccggtttc cgttatcggg gaagaagtgg ctgatctcag
     1501 ccaccgcgaa aatgacatca aaaacgccat taacctgatg ttctggggaa tatgaacggt
     1561 ctcgttccta atgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
     1621 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     1681 ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc
     1741 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     1801 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     1861 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     1921 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     1981 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     2041 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     2101 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     2161 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     2221 ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc
     2281 atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg
     2341 aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag
     2401 atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc
     2461 ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga
     2521 gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc
     2581 gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag
     2641 acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg
     2701 caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac
     2761 ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg
     2821 gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat
     2881 ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc
     2941 atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc
     3001 ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga
     3061 cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag
     3121 ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     3181 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     3241 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     3301 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     3361 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     3421 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     3481 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     3541 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     3601 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     3661 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     3721 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3781 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3841 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     3901 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     3961 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     4021 cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     4081 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
     4141 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     4201 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     4261 ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc
     4321 gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc
     4381 atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg
     4441 ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat
     4501 gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg
     4561 cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt
     4621 gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc
     4681 gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt
     4741 gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat
     4801 tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc
     4861 acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa
     4921 cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc
     4981 gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc
     5041 cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg
     5101 gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     5161 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     5221 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     5281 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     5341 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     5401 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     5461 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atgtccgcac caacgcgcag
     5521 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     5581 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     5641 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     5701 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     5761 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
     5821 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     5881 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     5941 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     6001 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     6061 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
     6121 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     6181 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     6241 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     6301 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     6361 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcg
//
