LOCUS       sequence-491746-        6405 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  GGA cloning his-mTagBFP2-AGGA-promoter-ccdb-TTCC.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6405)
  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 6405)
  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: cwby0011
FEATURES             Location/Qualifiers
     source          1..6405
                     /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             1247..1552
                     /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     1431..1450
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(1621..1639)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1635..1682
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1719..2174
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1806..1825)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2016..2037
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2266..3081)
                     /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(2266..3081)
                     /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     2989..3008
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3073..3092
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3203..3791
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3692..3711
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3945..3962
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3977..4116
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4042..4061)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4202..4224
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4218..4409)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5218..6300)
                     /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     6262..6281
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6301..6378)
                     /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 gtgagcaaag gcgaagaact
      421 gatcaaagaa aatatgcata tgaaactgta tatggaaggt accgtggata accatcattt
      481 taaatgcacc agcgaaggcg agggcaaacc gtatgaaggc acccagacca tgcgcattaa
      541 agtggtggaa ggcggcccgc tgccgtttgc gtttgatatt ctggcgacca gctttctgta
      601 tggcagcaaa acctttatta accataccca gggcattccg gatttcttta aacagagctt
      661 tccggaaggc tttacctggg aacgcgtgac cacctatgaa gatggcggcg tgctgaccgc
      721 gacccaggat accagcctgc aggatggctg cctgatttat aacgtgaaaa ttcgcggcgt
      781 gaactttacc agcaacggcc cggtgatgca gaagaaaacc ctgggctggg aagcgtttac
      841 cgaaaccctg tatccggcgg atggcggtct ggaaggccgc aacgatatgg cgctgaaact
      901 ggtgggcggt agccatctga ttgcgaacgc gaaaaccacc tatcgcagca agaaaccggc
      961 caagaacctg aaaatgccgg gcgtgtatta tgtggattat cgcctggaac gtattaaaga
     1021 agcgaacaac gaaacctatg tggaacagca tgaagtggcg gtggcgcgct attgcgatct
     1081 gccgagcaaa ctgggccata aactgaacgg cggcagcggc tctggtggct caggaggaga
     1141 ccgaaagcgt attacagtga cagttgacag cgacagctat cagttgctca aggcatatat
     1201 gatgtcaata tctccggtct ggtatgcaca accaagaatg aagcccatgc agtttaaggt
     1261 ttacacctat aaaagagaga gccgttatcg tctgtttgtg gatgtacaga gtgatattat
     1321 tgacacgccc gggcgacgga tggtgatccc cctggccagt gcacgtctgc tgtcagataa
     1381 agtctcccgt gaactttacc cggtggtgca tatcggggat gaaagctggc gcatgatgac
     1441 caccgatatg gccagtgtgc cggtttccgt tatcggggaa gaagtggctg atctcagcca
     1501 ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc tggggaatat gaacggtctc
     1561 gttcctaatg agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
     1621 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     1681 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
     1741 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     1801 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     1861 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     1921 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     1981 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     2041 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     2101 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     2161 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     2221 tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc
     2281 gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
     2341 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
     2401 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc
     2461 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
     2521 tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     2581 atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg
     2641 aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag
     2701 gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg
     2761 gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     2821 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc
     2881 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
     2941 gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     3001 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt
     3061 ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt
     3121 tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     3181 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     3241 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     3301 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     3361 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     3421 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     3481 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     3541 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     3601 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     3661 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     3721 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     3781 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     3841 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     3901 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     3961 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     4021 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     4081 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     4141 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     4201 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     4261 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     4321 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     4381 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     4441 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     4501 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     4561 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     4621 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     4681 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     4741 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     4801 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     4861 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
     4921 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
     4981 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
     5041 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
     5101 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     5161 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     5221 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     5281 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     5341 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     5401 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     5461 tgagctgtct tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc
     5521 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     5581 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     5641 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     5701 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     5761 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     5821 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     5881 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     5941 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     6001 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     6061 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     6121 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     6181 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     6241 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     6301 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     6361 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcg
//
