LOCUS       sequence-491751-        5673 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GGA cloning MS-his-AGGA-promoter-ccdb-TTCC.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5673)
  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 5673)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 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: cwby0005
FEATURES             Location/Qualifiers
     source          1..5673
                     /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             515..820
                     /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     699..718
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(889..907)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      903..950
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      987..1442
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1074..1093)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1284..1305
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1534..2349)
                     /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(1534..2349)
                     /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     2257..2276
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2341..2360
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2471..3059
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2960..2979
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3213..3230
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3245..3384
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3310..3329)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3470..3492
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3486..3677)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4486..5568)
                     /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     5530..5549
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5569..5646)
                     /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 accaccacca ccaccactca ggaggagacc gaaagcgtat
      421 tacagtgaca gttgacagcg acagctatca gttgctcaag gcatatatga tgtcaatatc
      481 tccggtctgg tatgcacaac caagaatgaa gcccatgcag tttaaggttt acacctataa
      541 aagagagagc cgttatcgtc tgtttgtgga tgtacagagt gatattattg acacgcccgg
      601 gcgacggatg gtgatccccc tggccagtgc acgtctgctg tcagataaag tctcccgtga
      661 actttacccg gtggtgcata tcggggatga aagctggcgc atgatgacca ccgatatggc
      721 cagtgtgccg gtttccgtta tcggggaaga agtggctgat ctcagccacc gcgaaaatga
      781 catcaaaaac gccattaacc tgatgttctg gggaatatga acggtctcgt tcctaatgag
      841 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
      901 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
      961 gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     1021 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     1081 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     1141 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     1201 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     1261 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     1321 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     1381 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     1441 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     1501 cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg
     1561 aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg
     1621 taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc
     1681 tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag
     1741 gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt
     1801 atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact
     1861 cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc
     1921 gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag
     1981 cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt
     2041 cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat
     2101 ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc
     2161 attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata
     2221 caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata
     2281 taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat
     2341 atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga
     2401 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     2461 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     2521 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2581 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     2641 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     2701 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     2761 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     2821 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     2881 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     2941 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     3001 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     3061 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     3121 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     3181 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     3241 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
     3301 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     3361 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     3421 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     3481 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     3541 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     3601 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     3661 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     3721 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     3781 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     3841 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     3901 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     3961 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     4021 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     4081 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     4141 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
     4201 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     4261 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
     4321 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     4381 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     4441 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     4501 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4561 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     4621 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     4681 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     4741 ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat
     4801 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     4861 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     4921 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     4981 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     5041 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     5101 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     5161 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     5221 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     5281 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     5341 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     5401 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     5461 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     5521 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     5581 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     5641 ggtgtccggg atctcgacgc tctcccttat gcg
//
