LOCUS       sequence-248524-        5762 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  coding sequence for synthetic zinc finger ZF-BCB-GCN.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5762)
  AUTHORS   Swank Z, Laohakunakorn N, Maerkl SJ
  TITLE     Cell-free gene-regulatory network engineering with synthetic 
            transcription factors.
  JOURNAL   Proc Natl Acad Sci U S A. 2019 Mar 26;116(13):5892-5901. doi: 
            10.1073/pnas.1816591116. Epub 2019 Mar 8.
  PUBMED    30850530
REFERENCE   2  (bases 1 to 5762)
  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"; doi: 
            "10.1073/pnas.1816591116"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2019-03-26- 26"; volume: "116"; issue: "13"; pages: 
            "5892-5901"
COMMENT     Sequence Label: pZF-BCB-GCN
FEATURES             Location/Qualifiers
     source          1..5762
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             62..118
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     primer_bind     complement(189..207)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      203..250
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      287..742
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(374..393)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     584..605
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        768..872
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(873..941,942..1733)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1091..1110)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1904..2492
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2393..2412
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2646..2663
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2678..2817
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2743..2762)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2903..2925
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2919..3110)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3919..5001)
                     /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     4963..4982
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5002..5079)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5285..5304
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5388..5407
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5388..5406
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5407..5431
                     /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             5446..5468
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 agcgccagcg ccaccgcatc gcgatattca gcatattctg ccgattctgg aagataaagt
       61 ggaagaactg ctgagcaaaa actatcatct ggaaaacgaa gtggcgcgcc tgaaaaaact
      121 ggtgggcgaa cgctaataag atccggctgc taacaaagcc cgaaaggaag ctgagttggc
      181 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
      241 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc
      301 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
      361 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
      421 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
      481 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
      541 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
      601 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
      661 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
      721 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
      781 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
      841 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
      901 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
      961 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     1021 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     1081 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     1141 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     1201 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     1261 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     1321 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     1381 ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca
     1441 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     1501 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     1561 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     1621 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     1681 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     1741 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     1801 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     1861 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     1921 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     1981 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     2041 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     2101 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     2161 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     2221 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     2281 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     2341 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     2401 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     2461 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     2521 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     2581 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     2641 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     2701 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     2761 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     2821 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     2881 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     2941 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     3001 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     3061 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     3121 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     3181 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     3241 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     3301 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     3361 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     3421 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     3481 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     3541 gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg
     3601 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     3661 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     3721 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     3781 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     3841 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     3901 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     3961 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     4021 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     4081 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     4141 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     4201 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     4261 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     4321 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     4381 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     4441 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     4501 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     4561 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     4621 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     4681 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     4741 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     4801 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     4861 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     4921 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     4981 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     5041 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     5101 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     5161 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     5221 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     5281 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     5341 ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac
     5401 tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa
     5461 gaaggagata tacatatgga acgtccgtac gcttgcccgg ttgaatcttg cgaccgtcgt
     5521 ttctctcgta acttcatcct gcagcgtcat attagaattc atactggaca aaaaccattc
     5581 caatgtagaa tttgtatgag aaatttctct cagcgttctt ctctggttcg tcacatccgt
     5641 acccacaccg gtgaaaaacc gttcgcttgc gacatctgcg gtcgtaaatt cgctcgtcac
     5701 gaccagctga cccgtcacac caaaatccac ctgcgtcaga aagacggtgg cggcggttct
     5761 cc
//
