LOCUS       sequence-248852-        5606 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  coding sequence for synthetic zinc finger ZF-AA-GCN.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5606)
  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 5606)
  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-AA-GCN
FEATURES             Location/Qualifiers
     source          1..5606
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     206..225
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     309..328
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        309..327
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    328..352
                     /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             367..389
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             589..645
                     /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(716..734)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      730..777
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      814..1269
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(901..920)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1111..1132
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1295..1399
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1400..1468,1469..2260)
                     /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(1618..1637)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2431..3019
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2920..2939
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3173..3190
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3205..3344
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3270..3289)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3430..3452
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3446..3637)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4446..5528)
                     /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     5490..5509
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5529..5606)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
       61 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
      121 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
      181 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
      241 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc
      301 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
      361 aataattttg tttaacttta agaaggagat atacatatga aaccgtttca gtgccgcatt
      421 tgcatgcgca actttagccg cagcgatcat ctgaccaccc atattcgcac ccataccggc
      481 gaaaaaccgt ttgcgtgcga tatttgcggc cgcaaattcg cccgcagcga tgaacgcaaa
      541 cgccatcgcg atattcagca tattctgccg attctggaag ataaagtgga agaactgctg
      601 agcaaaaact atcatctgga aaacgaagtg gcgcgcctga aaaaactggt gggcgaacgc
      661 taataagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
      721 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
      781 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
      841 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
      901 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagatc
      961 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     1021 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     1081 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     1141 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     1201 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     1261 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     1321 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     1381 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     1441 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     1501 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     1561 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     1621 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     1681 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     1741 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     1801 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     1861 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     1921 cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg
     1981 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     2041 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     2101 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     2161 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     2221 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     2281 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     2341 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     2401 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     2461 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     2521 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     2581 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     2641 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     2701 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     2761 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     2821 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     2881 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     2941 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     3001 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     3061 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     3121 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     3181 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     3241 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     3301 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     3361 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     3421 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     3481 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     3541 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     3601 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     3661 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     3721 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     3781 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     3841 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     3901 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     3961 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     4021 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     4081 ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc
     4141 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga
     4201 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa
     4261 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
     4321 gtgcggcgac gatagtcatg cccctcgccc accggaagga gctgactggg ttgaaggctc
     4381 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     4441 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4501 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     4561 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     4621 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     4681 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg
     4741 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     4801 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     4861 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     4921 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     4981 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     5041 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     5101 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     5161 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     5221 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     5281 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     5341 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     5401 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     5461 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     5521 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     5581 aaggttttgc gccattcgat ggtgtc
//
