LOCUS       sequence-491744-        6390 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  GGA cloning his-mScarletI-AGGA-promoter-ccdb-TTCC-his.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6390)
  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 6390)
  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: cwby0009
FEATURES             Location/Qualifiers
     source          1..6390
                     /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             1232..1537
                     /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     1416..1435
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(1606..1624)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1620..1667
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1704..2159
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1791..1810)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2001..2022
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2251..3066)
                     /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(2251..3066)
                     /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     2974..2993
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3058..3077
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3188..3776
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3677..3696
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3930..3947
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3962..4101
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4027..4046)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4187..4209
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4203..4394)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5203..6285)
                     /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     6247..6266
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6286..6363)
                     /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 gcgaagcggt
      421 gattaaagaa tttatgcgct ttaaagtgca tatggaaggc agcatgaacg gccatgaatt
      481 tgaaattgaa ggcgaaggtg aaggtcgccc gtatgaaggc acccagaccg cgaaactgaa
      541 agtgaccaaa ggcggcccgc tgccgtttag ctgggatatt ctgagcccgc agtttatgta
      601 tggcagccgc gcgtttatta aacatccggc ggatattccg gattattata aacagagctt
      661 tccggaaggt tttaaatggg aacgcgtgat gaactttgaa gatggcggcg cggtgaccgt
      721 gacccaggat accagcctgg aagatggcac cctgatttat aaagtgaaac tgcgcggcac
      781 caactttccg ccggatggcc cggtgatgca gaagaaaacc atgggctggg aagcgagcac
      841 cgaacgcctg tatccggaag atggtgtgct gaaaggtgat attaaaatgg cgctgcgcct
      901 gaaagatggc ggtcgctatc tggcggattt taaaaccacc tataaagcga agaaaccggt
      961 gcagatgccg ggcgcgtata acgtggatcg caaactggat atcaccagcc ataacgaaga
     1021 ttataccgtg gtggaacagt atgaacgcag cgaaggccgc catagcaccg gcggcatgga
     1081 tgaactgtat aaaggtggca gcggcagcgg tggctcagga ggagaccgaa agcgtattac
     1141 agtgacagtt gacagcgaca gctatcagtt gctcaaggca tatatgatgt caatatctcc
     1201 ggtctggtat gcacaaccaa gaatgaagcc catgcagttt aaggtttaca cctataaaag
     1261 agagagccgt tatcgtctgt ttgtggatgt acagagtgat attattgaca cgcccgggcg
     1321 acggatggtg atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact
     1381 ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag
     1441 tgtgccggtt tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat
     1501 caaaaacgcc attaacctga tgttctgggg aatatgaacg gtctcgttcc taatgagatc
     1561 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
     1621 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     1681 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     1741 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     1801 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     1861 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     1921 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     1981 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2041 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     2101 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     2161 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     2221 tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa
     2281 ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa
     2341 tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc
     2401 gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt
     2461 atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg
     2521 catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc
     2581 atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct
     2641 gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc
     2701 atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc
     2761 ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt
     2821 cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt
     2881 ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa
     2941 tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa
     3001 atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg
     3061 gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca
     3121 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     3181 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     3241 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     3301 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     3361 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     3421 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     3481 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     3541 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     3601 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     3661 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     3721 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     3781 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     3841 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     3901 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     3961 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac
     4021 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     4081 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     4141 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     4201 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     4261 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     4321 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     4381 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     4441 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     4501 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     4561 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     4621 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     4681 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     4741 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     4801 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt
     4861 ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca
     4921 gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc
     4981 atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc
     5041 tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc
     5101 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc
     5161 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag
     5221 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     5281 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt
     5341 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
     5401 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
     5461 atcgtcgtat cccactaccg agatgtccgc accaacgcgc agcccggact cggtaatggc
     5521 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
     5581 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
     5641 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
     5701 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
     5761 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat
     5821 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
     5881 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc
     5941 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
     6001 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
     6061 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
     6121 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
     6181 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga
     6241 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa
     6301 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt
     6361 gtccgggatc tcgacgctct cccttatgcg
//
