LOCUS       sequence-97724-e        5855 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A modified pET28 driving expression of GFP, biobrick part Bba_E0040.
            No LacO site downstream of PT7. T7 promoter has base pairs 'CCCT' 
            between -11 and -8..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5855)
  AUTHORS   Shis DL, Bennett MR
  TITLE     Library of synthetic transcriptional AND gates built with split T7 
            RNA polymerase mutants.
  JOURNAL   Proc Natl Acad Sci U S A. 2013 Mar 26;110(13):5028-33. doi: 
            10.1073/pnas.1220157110. Epub 2013 Mar 11.
  PUBMED    23479654
REFERENCE   2  (bases 1 to 5855)
  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.1220157110"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2013-03-26- 26"; volume: "110"; issue: "13"; pages: "5028-33"
COMMENT     Sequence Label: pET:CCCT:GFP
FEATURES             Location/Qualifiers
     source          1..5855
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(108..821)
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(148..168)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     763..791
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     complement(956..975)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1181..1258
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1259..2341
                     /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(1278..1297)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3150..3341
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3335..3357)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3443..3585
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3498..3517
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3600..3617)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3771..4359)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3851..3870)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4470..4489)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4481..5296
                     /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             4481..5296
                     /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     complement(4554..4573)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5389..5844)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5526..5547)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5738..5757
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagggcgcgc cttattattt gtatagttca
      121 tccatgccat gtgtaatccc agcagctgtt acaaactcaa gaaggaccat gtggtctctc
      181 ttttcgttgg gatctttcga aagggcagat tgtgtggaca ggtaatggtt gtctggtaaa
      241 aggacagggc catcgccaat tggagtattt tgttgataat ggtctgctag ttgaacgctt
      301 ccatcttcaa tgttgtgtct aattttgaag ttaactttga ttccattctt ttgtttgtct
      361 gccatgatgt atacattgtg tgagttatag ttgtattcca atttgtgtcc aagaatgttt
      421 ccatcttctt taaaatcaat accttttaac tcgattctat taacaagggt atcaccttca
      481 aacttgactt cagcacgtgt cttgtagttc ccgtcatctt tgaaaaatat agttctttcc
      541 tgtacataac cttcgggcat ggcactcttg aaaaagtcat gctgtttcat atgatctggg
      601 tatctcgcaa agcattgaac accataaccg aaagtagtga caagtgttgg ccatggaaca
      661 ggtagttttc cagtagtgca aataaattta agggtaagtt ttccgtatgt tgcatcacct
      721 tcaccctctc cactgacaga aaatttgtgc ccattaacat caccatctaa ttcaacaaga
      781 attgggacaa ctccagtgaa aagttcttct cctttacgca tgacgtcggt atatctcctt
      841 cttaaagtta aaccctatag tgaggggtat taatttcgcg ggatcgagat ctcgatcctc
      901 tacgccggac gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc tggcgcctat
      961 atcgccgaca tcaccgatgg ggaagatcgg gctcgccact tcgggctcat gagcgcttgt
     1021 ttcggcgtgg gtatggtggc aggccccgtg gccgggggac tgttgggcgc catctccttg
     1081 catgcaccat tccttgcggc ggcggtgctc aacggcctca acctactact gggctgcttc
     1141 ctaatgcagg agtcgcataa gggagagcgt cgagatcccg gacaccatcg aatggcgcaa
     1201 aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatgt
     1261 gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc
     1321 ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc
     1381 gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc
     1441 gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc
     1501 ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg
     1561 aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg
     1621 gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac
     1681 taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt
     1741 ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca
     1801 aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggctg
     1861 gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag
     1921 tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc
     1981 gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg
     2041 gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag acagctcatg
     2101 ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt
     2161 ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt
     2221 ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct ctccccgcgc
     2281 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     2341 agcgcaacgc aattaatgta agttagctca ctcattaggc accgggatct cgaccgatgc
     2401 ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg
     2461 ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct
     2521 gggtcatttt cggcgaggac cgctttcgct ggagcgcgac gatgatcggc ctgtcgcttg
     2581 cggtattcgg aatcttgcac gccctcgctc aagccttcgt cactggtccc gccaccaaac
     2641 gtttcggcga gaagcaggcc attatcgccg gcatggcggc cccacgggtg cgcatgatcg
     2701 tgctcctgtc gttgaggacc cggctaggct ggcggggttg ccttactggt tagcagaatg
     2761 aatcaccgat acgcgagcga acgtgaagcg actgctgctg caaaacgtct gcgacctgag
     2821 caacaacatg aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg cggaagtcag
     2881 cgccctgcac cattatgttc cggatctgca tcgcaggatg ctgctggcta ccctgtggaa
     2941 cacctacatc tgtattaacg aagcgctggc attgaccctg agtgattttt ctctggtccc
     3001 gccgcatcca taccgccagt tgtttaccct cacaacgttc cagtaaccgg gcatgttcat
     3061 catcagtaac ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt acccccatga
     3121 acagaaatcc cccttacacg gaggcatcag tgaccaaaca ggaaaaaacc gcccttaaca
     3181 tggcccgctt tatcagaagc cagacattaa cgcttctgga gaaactcaac gagctggacg
     3241 cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca
     3301 gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
     3361 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
     3421 cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt
     3481 atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc atatatgcgg
     3541 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc
     3601 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     3661 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     3721 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     3781 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     3841 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     3901 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     3961 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4021 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4081 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4141 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4201 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4261 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4321 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     4381 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgaacaat
     4441 aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata ttcaacggga
     4501 aacgtcttgc tctaggccgc gattaaattc caacatggat gctgatttat atgggtataa
     4561 atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc
     4621 cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga
     4681 tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt
     4741 tatccgtact cctgatgatg catggttact caccactgcg atccccggga aaacagcatt
     4801 ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt
     4861 cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt
     4921 tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga
     4981 tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata aacttttgcc
     5041 attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga
     5101 cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca
     5161 ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct
     5221 ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct
     5281 cgatgagttt ttctaagaat taattcatga gcggatacat atttgaatgt atttagaaaa
     5341 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa attgtaaacg
     5401 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     5461 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     5521 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     5581 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     5641 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     5701 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     5761 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     5821 ttaatgcgcc gctacagggc gcgtcccatt cgcca
//
