LOCUS       sequence-194390-        4614 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP under WT T7 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4614)
  AUTHORS   Abil Z, Ellefson JW, Gollihar JD, Watkins E, Ellington AD
  TITLE     Compartmentalized partnered replication for the directed evolution 
            of genetic parts and circuits.
  JOURNAL   Nat Protoc. 2017 Dec;12(12):2493-2512. doi: 10.1038/nprot.2017.119. 
            Epub 2017 Nov 9.
  PUBMED    29120463
REFERENCE   2  (bases 1 to 4614)
  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/nprot.2017.119"; journalName: "Nat Protoc"; date: 
            "2017-12"; volume: "12"; issue: "12"; pages: "2493-2512"
COMMENT     Sequence Label: pACYC-GFPM2
FEATURES             Location/Qualifiers
     source          1..4614
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(163..1245)
                     /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     1207..1226
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1246..1323)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1369..1388
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1369..1387
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1388..1412
                     /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)."
     CDS             1497..2213
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFAYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(1527..1555)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2150..2170
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2221..2268
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      2271..2318
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             2358..2402
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2440..2458)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2454..2501
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2722..3381)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3296..3315
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3382..3484)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(3873..3890)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4007..4552)
                     /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(4087..4106)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgtagcacct gaagtcagcc ccatacgata taagttgtaa ttctcatgtt agtcatgccc
       61 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc
      121 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag
      181 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
      241 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt
      301 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
      361 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
      421 atcgtcgtat cccactaccg agatgtccgc accaacgcgc agcccggact cggtaatggc
      481 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
      541 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
      601 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
      661 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
      721 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat
      781 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
      841 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc
      901 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
      961 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
     1021 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
     1081 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
     1141 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga
     1201 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa
     1261 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt
     1321 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaaatta atacgactca
     1381 ctatagggga attgtgagcg gataacaatt cccctgtaga aataattttg tttaacttta
     1441 attctagagt cacacaggac tactagatgg ggatgaccat gattacgcca agctttatga
     1501 gtaaaggaga agaacttttc actggagttg tcccaattct tgttgaatta gatggtgatg
     1561 ttaatgggca caaattttct gtcagtggag agggtgaagg tgatgcaaca tacggaaaac
     1621 ttacccttaa atttatttgc actactggaa aactacctgt tccatggcca acacttgtca
     1681 ctactttcgc gtatggtctt caatgctttg cgagataccc agatcatatg aaacagcatg
     1741 actttttcaa gagtgccatg cccgaaggtt atgtacagga aagaactata tttttcaaag
     1801 atgacgggaa ctacaagaca cgtgctgaag tcaaatttga aggtgatacc cttgttaata
     1861 gaatcgagtt aaaaggtatt gattttaaag aagatggaaa cattcttgga cacaaattgg
     1921 aatacaacta taactcacac aatgtataca tcatggcaga caaacaaaag aatggaatca
     1981 aagttaactt caaaattaga cacaacattg aagatggaag cgttcaacta gcagaccatt
     2041 atcaacaaaa tactccaatt ggcgatggcc ctgtcctttt accagacaac cattacctgt
     2101 ccacacaatc tgccctttcg aaagatccca acgaaaagag agaccacatg gtccttcttg
     2161 agtttgtaac agctgctggg attacacatg gcatggatga actatacaaa tagaattaga
     2221 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgca ctagcataac
     2281 cccttggggc ctctaaacgg gtcttgaggg gttttttgcg aattcgatct caattggata
     2341 ttccactcga gtctggtaaa gaaaccgctg ctgcgaaatt tgaacgccag cacatggact
     2401 cgtctactag cgcagcttaa ttaacctagg ctgctgccac cgctgagcaa taactagcat
     2461 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaacct caggcatttg
     2521 agaagcacac ggtcacactg cttccggtag tcaataaacc ggtaaaccag caatagacat
     2581 aagcggctat ttaacgaccc tgccctgaac cgacgaccgg gtcgaatttg ctttcgaatt
     2641 tctgccattc atccgcttat tatcacttat tcaggcgtag caaccaggcg tttaagggca
     2701 ccaataactg ccttaaaaaa attacgcccc gccctgccac tcatcgcagt actgttgtaa
     2761 ttcattaagc attctgccga catggaagcc atcacaaacg gcatgatgaa cctgaatcgc
     2821 cagcggcatc agcaccttgt cgccttgcgt ataatatttg cccatagtga aaacgggggc
     2881 gaagaagttg tccatattgg ccacgtttaa atcaaaactg gtgaaactca cccagggatt
     2941 ggctgagacg aaaaacatat tctcaataaa ccctttaggg aaataggcca ggttttcacc
     3001 gtaacacgcc acatcttgcg aatatatgtg tagaaactgc cggaaatcgt cgtggtattc
     3061 actccagagc gatgaaaacg tttcagtttg ctcatggaaa acggtgtaac aagggtgaac
     3121 actatcccat atcaccagct caccgtcttt cattgccata cggaactccg gatgagcatt
     3181 catcaggcgg gcaagaatgt gaataaaggc cggataaaac ttgtgcttat ttttctttac
     3241 ggtctttaaa aaggccgtaa tatccagctg aacggtctgg ttataggtac attgagcaac
     3301 tgactgaaat gcctcaaaat gttctttacg atgccattgg gatatatcaa cggtggtata
     3361 tccagtgatt tttttctcca ttttagcttc cttagctcct gaaaatctcg ataactcaaa
     3421 aaatacgccc ggtagtgatc ttatttcatt atggtgaaag ttggaacctc ttacgtgccg
     3481 atcacgtctc attttcgcca aagttggcca gggcttcccg gtatcaacag ggacaccagg
     3541 atttatttat nntgcgaagt gatcttccgt cacaggtatt tattcggcgc aaagtgcgtc
     3601 gggtgatgct gccaacttac tgatttagtg tatgatggtg tttttgaggt gctccagtgg
     3661 cttctgtttc tatcagctgt ccctcctgtt cagctactga cggggtggtg cgtaacggca
     3721 aaagcaccgc cggacatcag cgctagcgga gtgtatactg gcttactatg ttggcactga
     3781 tgagggtgtc agtgaagtgc ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag
     3841 cagaatatgt gatacaggat atattccgct tcctcgctca ctgactcgct acgctcggtc
     3901 gttcgactgc ggcgagcgga aatggcttac gaacggggcg gagatttcct ggaagatgcc
     3961 aggaagatac ttaacaggga agtgagaggg ccgcggcaaa gccgtttttc cataggctcc
     4021 gcccccctga caagcatcac gaaatctgac gctcaaatca gtggtggcga aacccgacag
     4081 gactataaag ataccaggcg tttccccctg gcggctccct cgtgcgctct cctgttcctg
     4141 cctttcggtt taccggtgtc attccgctgt tatggccgcg tttgtctcat tccacgcctg
     4201 acactcagtt ccgggtaggc agttcgctcc aagctggact gtatgcacga accccccgtt
     4261 cagtccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggaaagacat
     4321 gcaaaagcac cactggcagc agccactggt aattgattta gaggagttag tcttgaagtc
     4381 atgcgccggt taaggctaaa ctgaaaggac aagttttggt gactgcgctc ctccaagcca
     4441 gttacctcgg ttcaaagagt tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc
     4501 ggttttttcg ttttcagagc aagagattac gcgcagacca aaacgatctc aagaagatca
     4561 tcttattaat cagataaaat atttctagat ttcagtgcaa tttatctctt caaa
//
