LOCUS       sequence-248906-        5762 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  coding sequence for synthetic zinc finger ZF-ADD-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-ADD-GCN
FEATURES             Location/Qualifiers
     source          1..5762
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     41..60
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        41..59
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    60..84
                     /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             99..121
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             477..533
                     /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(604..622)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      618..665
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      702..1157
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(789..808)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     999..1020
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1183..1287
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1288..1356,1357..2148)
                     /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(1506..1525)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2319..2907
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2808..2827
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3061..3078
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3093..3232
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3158..3177)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3318..3340
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3334..3525)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4334..5416)
                     /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     5378..5397
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5417..5494)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5700..5719
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg
       61 gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag
      121 atatacatat ggaacgtccg tacgcttgcc cggttgaatc ttgcgaccgt cgtttctctg
      181 acaaaaccaa actgcgtgtt catattagaa ttcatactgg acaaaaacca ttccaatgta
      241 gaatttgtat gagaaatttc tctgttcgtc acaacctgac ccgtcacatc cgtacccaca
      301 ccggtgaaaa accgttcgct tgcgacatct gcggtcgtaa attcgctcgt tctgacgaac
      361 gtaaacgtca caccaaaatc cacctgcgtc agaaagacgg tggcggcggt tctccagcgc
      421 cagcgccacc gcatcgcgat attcagcata ttctgccgat tctggaagat aaagtggaag
      481 aactgctgag caaaaactat catctggaaa acgaagtggc gcgcctgaaa aaactggtgg
      541 gcgaacgcta ataagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
      601 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
      661 ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc
      721 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
      781 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
      841 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
      901 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
      961 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     1021 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     1081 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     1141 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     1201 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     1261 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     1321 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     1381 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     1441 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     1501 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     1561 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     1621 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     1681 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     1741 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     1801 ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta
     1861 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     1921 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     1981 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     2041 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     2101 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     2161 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     2221 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     2281 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     2341 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     2401 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     2461 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     2521 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     2581 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     2641 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     2701 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     2761 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     2821 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     2881 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     2941 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     3001 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     3061 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     3121 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     3181 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     3241 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     3301 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     3361 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     3421 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     3481 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     3541 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     3601 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     3661 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     3721 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     3781 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     3841 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     3901 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     3961 caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg
     4021 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
     4081 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
     4141 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac
     4201 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     4261 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     4321 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     4381 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     4441 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     4501 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     4561 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg
     4621 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     4681 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     4741 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     4801 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     4861 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     4921 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
     4981 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
     5041 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
     5101 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
     5161 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
     5221 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
     5281 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
     5341 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
     5401 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
     5461 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc
     5521 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
     5581 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
     5641 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
     5701 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
     5761 at
//
