LOCUS       sequence-73355-e        7399 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7399)
  AUTHORS   Stanton BC, Nielsen AA, Tamsir A, Clancy K, Peterson T, Voigt CA
  TITLE     Genomic mining of prokaryotic repressors for orthogonal logic gates.
  JOURNAL   Nat Chem Biol. 2013 Dec 8. doi: 10.1038/nchembio.1411.
  PUBMED    24316737
REFERENCE   2  (bases 1 to 7399)
  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"; journalName: "Nat Chem 
            Biol. 2013 Dec 8. doi: 10.1038/nchembio.1411."
COMMENT     Sequence Label: pNAND-PhlF
FEATURES             Location/Qualifiers
     source          1..7399
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(132..149)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(266..811)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(346..365)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     883..902
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(1877..2959)
                     /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     2921..2940
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(3094..3099,3100..3115,3116..3122)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    3128..3147
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     terminator      3807..3878
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3894..3921
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     RBS             4080..4091
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(4098..4100,4101..4103,4104..4817)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4143..4164)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4404..4423)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4751..4772
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4857..4874)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      5027..5113
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5205..5232
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        5251..5342
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5343..5411,5412..6203)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      6245..6700
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6332..6351)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gctagcggag tgtatactgg cttactatgt tggcactgat gagggtgtca gtgaagtgct
       61 tcatgtggca ggagaaaaaa ggctgcaccg gtgcgtcagc agaatatgtg atacaggata
      121 tattccgctt cctcgctcac tgactcgcta cgctcggtcg ttcgactgcg gcgagcggaa
      181 atggcttacg aacggggcgg agatttcctg gaagatgcca ggaagatact taacagggaa
      241 gtgagagggc cgcggcaaag ccgtttttcc ataggctccg cccccctgac aagcatcacg
      301 aaatctgacg ctcaaatcag tggtggcgaa acccgacagg actataaaga taccaggcgt
      361 ttccccctgg cggctccctc gtgcgctctc ctgttcctgc ctttcggttt accggtgtca
      421 ttccgctgtt atggccgcgt ttgtctcatt ccacgcctga cactcagttc cgggtaggca
      481 gttcgctcca agctggactg tatgcacgaa ccccccgttc agtccgaccg ctgcgcctta
      541 tccggtaact atcgtcttga gtccaacccg gaaagacatg caaaagcacc actggcagca
      601 gccactggta attgatttag aggagttagt cttgaagtca tgcgccggtt aaggctaaac
      661 tgaaaggaca agttttggtg actgcgctcc tccaagccag ttacctcggt tcaaagagtt
      721 ggtagctcag agaaccttcg aaaaaccgcc ctgcaaggcg gttttttcgt tttcagagca
      781 agagattacg cgcagaccaa aacgatctca agaagatcat cttattaatc agataaaata
      841 tttgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
      901 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
      961 gatctgctca tgtttgacag cttatcatcg atgcataatg tgcctgtcaa atggacgaag
     1021 cagggattct gcaaacccta tgctactccc tcgagccgtc aattgtctga ttcgttacca
     1081 attattaatt tttaaagtat gggcaatcaa ttgctcctgt taaaattgct ttagaaatac
     1141 tttggcagcg gtttgttgta ttgagtttca tttgcgcatt ggttaaatgg aaagtgacag
     1201 tacgctcact gcaacctaat atttttgaaa tatcccaaga gctttttcct tcgcatgccc
     1261 acgctaaaca ttctttttct cttttggtta aatcgttgtt tgatttatta tttgctatat
     1321 ttatttttcg ataattatca actagagaag gaacaattaa tggtatgttc atacacgcat
     1381 gtaaaaataa actatctata tagttgtctt tttctgaatg tgcaaaacta agcattccga
     1441 agccattgtt agccgtatga atagggaaac taaacccagt gataagacct gatgttttcg
     1501 cttctttaat tacatttgga gattttttat ttacagcatt gttttcaaat atattccaat
     1561 taattggtga atgattggag ttagaataat ctactatagg atcatatttt attaaattag
     1621 cgtcatcata atattgcctc cattttttag ggtaattatc taggattgaa atatcagatt
     1681 taaccataga atgaggataa atgatcgcga gtaaataata ttcacaatgt accattttag
     1741 tcatatcaga taagcattga ttaatatcat tattgcttct acaagcttta attttattaa
     1801 ttattctgta tgtgtcgtcg gcatttatgt ttttcatcga ggcctttcct gtgtgacccg
     1861 ctggaatagg atcttatcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     1921 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     1981 ttcttttcac cagtgagact ggcaacagct gattgccctt caccgcctgg ccctgagaga
     2041 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     2101 ttaacggcgg gatataacat gagctatctt cggtatcgtc gtatcccact accgagatat
     2161 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     2221 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     2281 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     2341 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     2401 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     2461 taccgtcctc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     2521 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     2581 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     2641 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     2701 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     2761 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     2821 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     2881 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     2941 ataacgttac tggtttcata ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     3001 ccataccgcg aaaggttttg cgccattcga tggcgcgccg cttcgtcagg ccacatagct
     3061 ttcttgttct gatcggaacg atcgttggct gtgttgacaa ttaatcatcg gctcgtataa
     3121 tgtgtggaat tgtgagcgct cacaattcta tggactatgt ttgaaaggga gaaatactag
     3181 atggcacgta ccccgagccg tagcagcatt ggtagcctgc gtagtccgca tacccataaa
     3241 gcaattctga ccagcaccat tgaaatcctg aaagaatgtg gttatagcgg tctgagcatt
     3301 gaaagcgttg cacgtcgtgc cggtgcaagc aaaccgacca tttatcgttg gtggaccaat
     3361 aaagcagcac tgattgccga agtgtatgaa aatgaaagcg aacaggtgcg taaatttccg
     3421 gatctgggta gctttaaagc cgatctggat tttctgctgc gtaatctgtg gaaagtttgg
     3481 cgtgaaacca tttgtggtga agcatttcgt tgtgttattg cagaagcaca gctggaccct
     3541 gcaaccctga cccagctgaa agatcagttt atggaacgtc gtcgtgagat gccgaaaaaa
     3601 ctggttgaaa atgccattag caatggtgaa ctgccgaaag ataccaatcg tgaactgctg
     3661 ctggatatga tttttggttt ttgttggtat cgcctgctga ccgaacagct gaccgttgaa
     3721 caggatattg aagaatttac cttcctgctg attaatggtg tttgtccggg tacacagcgt
     3781 taactagggc ccataccccc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg
     3841 cctttcgttt tatctgttgt ttgtcggtga acgctctcta ctagagtcac actggctcac
     3901 cttcgggtgg gcctttctgc gtttatacga cgtacggtgg aatctgattc gttaccaatt
     3961 gacatgatac gaaacgtacc gtatcgttaa ggtcgctcat tcactaggtc tgattcgtta
     4021 ccaattgaca actggtggtc gaatcaagat aatagaccag tcactatatt ttactagaga
     4081 aagaggagaa atactagatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     4141 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     4201 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     4261 ccgtgccctg gcccaccctc gtgaccacct tcggctacgg cctgcaatgc ttcgcccgct
     4321 accccgacca catgaagctg cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     4381 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     4441 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     4501 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     4561 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     4621 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     4681 tgctgcccga caaccactac ctgagctacc agtccgccct gagcaaagac cccaacgaga
     4741 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     4801 acgagctgta caagtaataa gcttggctgt tttggcggat gagagaagat tttcagcctg
     4861 atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc tggcggcagt
     4921 agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg tagcgccgat
     4981 ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa
     5041 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct
     5101 gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg
     5161 gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg ccatcctgac
     5221 ggatggcctt tttgcgtttc tacaaactct tttgtttatt tttctaaata cattcaaata
     5281 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     5341 ctatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     5401 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     5461 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     5521 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     5581 cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     5641 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     5701 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     5761 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     5821 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     5881 tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     5941 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     6001 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     6061 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     6121 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     6181 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     6241 atttacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     6301 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     6361 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     6421 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
     6481 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     6541 agtggactct tgttccaaac ttgaacaaca ctcaacccta tctcgggcta ttcttttgat
     6601 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     6661 tttaacgcga attttaacaa aatattaacg tttacaattt aaaaggatct aggtgaagat
     6721 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     6781 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     6841 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     6901 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     6961 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     7021 cgctctgcta atcctgttac cagtcaggca tttgagaagc acacggtcac actgcttccg
     7081 gtagtcaata aaccggtaaa ccagcaatag acataagcgg ctatttaacg accctgccct
     7141 gaaccgacga ccgggtcgaa tttgctttcg aatttctgcc attcatccgc ttattatcac
     7201 ttattcaggc gtagcaccag gcgtttaagg gcaccaataa ctgccttaaa aaaattacgc
     7261 cccgccctgc cactcatcgc agtactgttg taattcatta agcattctgc cgacatggaa
     7321 gccatcacag acggcatgat gaacctgaat cgccagcggc atcagcacct tgtcgccttg
     7381 cgtataatat ttgcccatg
//
