LOCUS       sequence-97359-e        7764 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  used with pSpec  to generate an AND gate without memory .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7764)
  AUTHORS   Yang L, Nielsen AA, Fernandez-Rodriguez J, McClune CJ, Laub MT, Lu 
            TK, Voigt CA
  TITLE     Permanent genetic memory with >1-byte capacity.
  JOURNAL   Nat Methods. 2014 Dec;11(12):1261-6. doi: 10.1038/nmeth.3147. Epub 
            2014 Oct 26.
  PUBMED    25344638
REFERENCE   2  (bases 1 to 7764)
  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: 
            "10.1038/nmeth.3147"; journalName: "Nat Methods"; date: "2014-12"; 
            volume: "11"; issue: "12"; pages: "1261-6"
COMMENT     Sequence Label: pAND_yfp
FEATURES             Location/Qualifiers
     source          1..7764
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(308..852)
                     /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(388..407)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(941..1756)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(941..1756)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1664..1683
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1748..1767
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    1881..1899
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1906..1924
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      2715..2786
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2802..2829
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        join(2842..2847,2848..2863,2864..2870)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2876..2895
                     /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      3602..3673
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3689..3716
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      4614..4685
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4701..4728
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     RBS             4813..4824
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(4831..4833,4834..4836,4837..5550)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4876..4897)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5137..5156)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5484..5505
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     terminator      5574..5645
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5661..5688
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             5781..6863
                     /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     complement(5800..5819)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             6882..7505
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(6955..6974)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(7688..7707)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gcttcctcgc tcactgactc gctgcacgag gcagacctca gcgctagcgg agtgtatact
       61 ggcttactat gttggcactg atgagggtgt cagtgaagtg cttcatgtgg caggagaaaa
      121 aaggctgcac cggtgcgtca gcagaatatg tgatacagga tatattccgc ttcctcgctc
      181 actgactcgc tacgctcggt cgttcgactg cggcgagcgg aaatggctta cgaacggggc
      241 ggagatttcc tggaagatgc caggaagata cttaacaggg aagtgagagg gccgcggcaa
      301 agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga cgctcaaatc
      361 agtggtggcg aaacccgaca ggactataaa gataccaggc gtttcccctg gcggctccct
      421 cgtgcgctct cctgttcctg cctttcggtt taccggtgtc attccgctgt tatggccgcg
      481 tttgtctcat tccacgcctg acactcagtt ccgggtaggc agttcgctcc aagctggact
      541 gtatgcacga accccccgtt cagtccgacc gctgcgcctt atccggtaac tatcgtcttg
      601 agtccaaccc ggaaagacat gcaaaagcac cactggcagc agccactggt aattgattta
      661 gaggagttag tcttgaagtc atgcgccggt taaggctaaa ctgaaaggac aagttttggt
      721 gactgcgctc ctccaagcca gttacctcgg ttcaaagagt tggtagctca gagaaccttc
      781 gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc aagagattac gcgcagacca
      841 aaacgatctc aagaagatca tcttattaag gggtctgacg ctcagtggaa cgaaaaatca
      901 atctaaagta tatatgagta aacttggtct gacagttacc ttagaaaaac tcatcgagca
      961 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
     1021 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
     1081 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
     1141 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
     1201 aaagcttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
     1261 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata
     1321 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
     1381 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
     1441 ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
     1501 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
     1561 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
     1621 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     1681 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctcgagcaa gacgtttccc
     1741 gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg
     1801 ttcatgatga tatattttta tcttgtgcaa tgtacatcag agattttgag acacaagctt
     1861 tactccaccg ttggcttttt tccctatcag tgatagagat tgacatccct atcagtgata
     1921 gagataatga gcacagatgc tgtagtggga tgtgtgtctc acctgaagag tacaaaagtc
     1981 cgaaacggta tcctctacaa ataattttgt ttaactatgg actatgtttt cacacaggaa
     2041 ataccaggat ggcacgtaaa accgcagcag aagcagaaga aacccgtcag cgtattattg
     2101 atgcagcact ggaagttttt gttgcacagg gtgttagtga tgcaaccctg gatcagattg
     2161 cacgtaaagc cggtgttacc cgtggtgcag tttattggca ttttaatggt aaactggaag
     2221 ttctgcaggc agttctggca agccgtcagc atccgctgga actggatttt acaccggatc
     2281 tgggtattga acgtagctgg gaagcagttg ttgttgcaat gctggatgca gttcatagtc
     2341 cgcagagcaa acagtttagc gaaattctga tttatcaggg tctggatgaa agcggtctga
     2401 ttcataatcg tatggttcag gcaagcgatc gttttctgca gtatattcat caggttctgc
     2461 gtcatgcagt tacccagggt gaactgccga ttaatctgga tctgcagacc agcattggtg
     2521 tttttaaagg tctgattacc ggtctgctgt atgaaggtct gcgtagcaaa gatcagcagg
     2581 cacagattat caaagttgca ctgggtagct tttgggcact gctgcgtgaa ccgcctcgtt
     2641 ttctgctgtg tgaagaagca cagattaaac aggtgaaatc cttcgaataa ctagggccca
     2701 taccccccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta
     2761 tctgttgttt gtcggtgaac gctctctact agagtcacac tggctcacct tcgggtgggc
     2821 ctttctgcgt ttatacgctt gttgacaatt aatcatcggc tcgtataatg tgtggaattg
     2881 tgagcgctca caattagact gtcgccggat gtgtatccga cctgacgatg gcccaaaagg
     2941 gccgaaacag tcctctacaa ataattttgt ttaactatgg actatgtttt aactactaga
     3001 tggaaagcac cccgaccaaa cagaaagcaa tttttagcgc aagcctgctg ctgtttgcag
     3061 aacgtggttt tgatgcaacc accatgccga tgattgcaga aaatgcaaaa gttggtgcag
     3121 gcaccattta tcgctatttc aaaaacaaag aaagcctggt gaacgaactg tttcagcagc
     3181 atgttaatga atttctgcag tgtattgaaa gcggtctggc aaatgaacgt gatggttatc
     3241 gtgatggctt tcatcacatt tttgaaggta tggtgacctt taccaaaaat catccgcgtg
     3301 cactgggttt tatcaaaacc catagccagg gcacctttct gaccgaagaa agccgtctgg
     3361 catatcagaa actggttgaa tttgtgtgca ccttttttcg tgaaggtcag aaacagggtg
     3421 tgattcgtaa tctgccggaa aatgcactga ttgcaattct gtttggcagc tttatggaag
     3481 tgtatgaaat gatcgagaac gattatctga gcctgaccga tgaactgctg accggtgttg
     3541 aagaaagcct gtgggcagca ctgagccgtc agagctaact agggcccata cccccaggca
     3601 tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc
     3661 ggtgaacgct ctctactaga gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta
     3721 taggagtcta tgattggtcc agattcgtta ccaattgaca gctagctcag tcctaggtat
     3781 atacatacat gcttgtttgt ttgtaaacaa tccgcgtgat aggtctgatt cgttaccaat
     3841 tgacggaatg aacgttcatt ccgataatgc tagcagtacg tctgagcgtg atacccgctc
     3901 actgaagatg gcccggtagg gccgaaacgt acctctacaa ataattttgt ttaactatgg
     3961 actatgtttg aaagggagaa atactagatg gcacgtaccc cgagccgtag cagcattggt
     4021 agcctgcgta gtccgcatac ccataaagca attctgacca gcaccattga aatcctgaaa
     4081 gaatgtggtt atagcggtct gagcattgaa agcgttgcac gtcgtgccgg tgcaagcaaa
     4141 ccgaccattt atcgttggtg gaccaataaa gcagcactga ttgccgaagt gtatgaaaat
     4201 gaaagcgaac aggtgcgtaa atttccggat ctgggtagct ttaaagccga tctggatttt
     4261 ctgctgcgta atctgtggaa agtttggcgt gaaaccattt gtggtgaagc atttcgttgt
     4321 gttattgcag aagcacagct ggaccctgca accctgaccc agctgaaaga tcagtttatg
     4381 gaacgtcgtc gtgagatgcc gaaaaaactg gttgaaaatg ccattagcaa tggtgaactg
     4441 ccgaaagata ccaatcgtga actgctgctg gatatgattt ttggtttttg ttggtatcgc
     4501 ctgctgaccg aacagctgac cgttgaacag gatattgaag aatttacctt cctgctgatt
     4561 aatggtgttt gtccgggtac acagcgttaa ctagggccca tacccccagg catcaaataa
     4621 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg
     4681 ctctctacta gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tatatacgcg
     4741 acgtacggtg gaatctgatt cgttaccaat tgacatgata cgaaacgtac cgtatcgtta
     4801 aggttactag agaaagagga gaaatactag atggtgagca agggcgagga gctgttcacc
     4861 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     4921 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     4981 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccttcggcta cggcctgcaa
     5041 tgcttcgccc gctaccccga ccacatgaag ctgcacgact tcttcaagtc cgccatgccc
     5101 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     5161 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     5221 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     5281 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     5341 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     5401 gacggccccg tgctgctgcc cgacaaccac tacctgagct accagtccgc cctgagcaaa
     5461 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     5521 actctcggca tggacgagct gtacaagtaa ctagggccca tacccccagg catcaaataa
     5581 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg
     5641 ctctctacta gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tataaatggc
     5701 ggcgcgccat cgaatggcgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga
     5761 gtcaattcag ggtggtgaat atgaaaccag taacgttata cgatgtcgca gagtatgccg
     5821 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa
     5881 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac
     5941 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc
     6001 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg
     6061 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc
     6121 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca
     6181 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga
     6241 cacccatcaa cagtattatt ttctcccatg aggacggtac gcgactgggc gtggagcatc
     6301 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg
     6361 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag
     6421 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga
     6481 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa
     6541 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg
     6601 acgataccga agatagctca tgttatatcc cgccgttaac caccatcaaa caggattttc
     6661 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
     6721 agggcaatca gctgttgcca gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
     6781 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     6841 cccgactgga aagcgggcag tgataatcca ggaggaaaaa aatgtccaga ttagataaaa
     6901 gtaaagtgat taacagcgca ttagagctgc ttaatgaggt cggaatcgaa ggtttaacaa
     6961 cccgtaaact cgcccagaag ctaggtgtag agcagcctac attgtattgg catgtaaaaa
     7021 ataagcgggc tttgctcgac gccttagcca ttgagatgtt agataggcac catactcact
     7081 tttgcccttt agaaggggaa agctggcaag attttttacg taataacgct aaaagtttta
     7141 gatgtgcttt actaagtcat cgcgatggag caaaagtaca tttaggtaca cggcctacag
     7201 aaaaacagta tgaaactctc gaaaatcaat tagccttttt atgccaacaa ggtttttcac
     7261 tagagaatgc attatatgca ctcagcgctg tggggcattt tactttaggt tgcgtattgg
     7321 aagatcaaga gcatcaagtc gctaaagaag aaagggaaac acctactact gatagtatgc
     7381 cgccattatt acgacaagct atcgaattat ttgatcacca aggtgcagag ccagccttct
     7441 tattcggcct tgaattgatc atatgcggat tagaaaaaca acttaaatgt gaaagtgggt
     7501 cctaataatt ggtaacgaat cagacaattg acggctcgag ggagtagcat agggtttgca
     7561 gaatccctgc ttcgtccatt tgacaggcac attatgcatc gatgataagc tgtcaaacat
     7621 gagcagatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt
     7681 gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc
     7741 atgagcaaat attttatctg aggt
//
