LOCUS       sequence-491745-        5642 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  GGA cloning TATG-promotor-ccdb-TAAT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5642)
  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 5642)
  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: cwby0006
FEATURES             Location/Qualifiers
     source          1..5642
                     /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             488..793
                     /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     672..691
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(858..876)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      872..919
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      956..1411
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1043..1062)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1253..1274
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1503..2318)
                     /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(1503..2318)
                     /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     2226..2245
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2310..2329
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2440..3028
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2929..2948
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3182..3199
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3214..3353
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3279..3298)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3439..3461
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3455..3646)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4455..5537)
                     /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     5499..5518
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5538..5615)
                     /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 catatgggag accgaaagcg tattacagtg acagttgaca gcgacagcta
      421 tcagttgctc aaggcatata tgatgtcaat atctccggtc tggtatgcac aaccaagaat
      481 gaagcccatg cagtttaagg tttacaccta taaaagagag agccgttatc gtctgtttgt
      541 ggatgtacag agtgatatta ttgacacgcc cgggcgacgg atggtgatcc ccctggccag
      601 tgcacgtctg ctgtcagata aagtctcccg tgaactttac ccggtggtgc atatcgggga
      661 tgaaagctgg cgcatgatga ccaccgatat ggccagtgtg ccggtttccg ttatcgggga
      721 agaagtggct gatctcagcc accgcgaaaa tgacatcaaa aacgccatta acctgatgtt
      781 ctggggaata tgaacggtct cgtaatgaga tccggctgct aacaaagccc gaaaggaagc
      841 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
      901 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg
      961 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     1021 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     1081 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     1141 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     1201 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     1261 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     1321 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     1381 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     1441 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat
     1501 tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     1561 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     1621 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     1681 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     1741 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     1801 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     1861 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     1921 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     1981 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca
     2041 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     2101 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     2161 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     2221 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     2281 ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt
     2341 atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca
     2401 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     2461 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     2521 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     2581 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     2641 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     2701 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     2761 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     2821 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     2881 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     2941 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     3001 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     3061 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     3121 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     3181 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     3241 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     3301 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     3361 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     3421 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     3481 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     3541 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     3601 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     3661 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     3721 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     3781 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     3841 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     3901 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     3961 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     4021 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     4081 tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct
     4141 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
     4201 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     4261 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     4321 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     4381 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     4441 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     4501 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     4561 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     4621 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     4681 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc
     4741 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     4801 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     4861 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     4921 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     4981 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     5041 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     5101 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     5161 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     5221 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     5281 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     5341 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     5401 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     5461 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     5521 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     5581 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     5641 cg
//
