LOCUS       sequence-248525-        5607 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  coding sequence for synthetic zinc finger ZF-ADD.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5607)
  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 5607)
  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
FEATURES             Location/Qualifiers
     source          1..5607
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1..23
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(347..365)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      361..408
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      445..900
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(532..551)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     742..763
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        926..1030
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1031..1099,1100..1891)
                     /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(1249..1268)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2062..2650
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2551..2570
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2804..2821
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2836..2975
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2901..2920)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3061..3083
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3077..3268)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4077..5159)
                     /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     5121..5140
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5160..5237)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5443..5462
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5550..5569
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5550..5568
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5569..5593
                     /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)."
ORIGIN
        1 tttgtttaac tttaagaagg agatatacat atggaacgtc cgtacgcttg cccggttgaa
       61 tcttgcgacc gtcgtttctc tgacaaaacc aaactgcgtg ttcatattag aattcatact
      121 ggacaaaaac cattccaatg tagaatttgt atgagaaatt tctctgttcg tcacaacctg
      181 acccgtcaca tccgtaccca caccggtgaa aaaccgttcg cttgcgacat ctgcggtcgt
      241 aaattcgctc gttctgacga acgtaaacgt cacaccaaaa tccacctgcg tcagtaagat
      301 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
      361 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
      421 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
      481 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
      541 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
      601 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
      661 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
      721 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
      781 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
      841 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
      901 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
      961 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     1021 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     1081 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     1141 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     1201 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     1261 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     1321 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     1381 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     1441 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     1501 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     1561 caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     1621 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     1681 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     1741 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     1801 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     1861 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     1921 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     1981 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     2041 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     2101 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     2161 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     2221 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     2281 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     2341 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     2401 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     2461 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     2521 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     2581 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     2641 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     2701 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     2761 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     2821 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     2881 ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     2941 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     3001 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     3061 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     3121 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
     3181 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     3241 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     3301 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     3361 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     3421 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     3481 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     3541 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     3601 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     3661 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     3721 tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
     3781 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
     3841 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
     3901 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     3961 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     4021 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     4081 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4141 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     4201 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     4261 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     4321 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
     4381 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     4441 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     4501 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     4561 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     4621 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     4681 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     4741 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     4801 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     4861 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     4921 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     4981 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     5041 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     5101 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     5161 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     5221 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     5281 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     5341 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     5401 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     5461 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     5521 gatcgagatc gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
     5581 ggataacaat tcccctctag aaataat
//
