LOCUS       sequence-199528-        4617 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 4617)
  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 4617)
  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..4617
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     213..233
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      284..331
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      334..381
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             421..465
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(503..521)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      517..564
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(785..1444)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1359..1378
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1445..1547)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(1939..1956)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2073..2618)
                     /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(2153..2172)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2843..3925)
                     /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     3887..3906
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3926..4003)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4049..4068
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4049..4067
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4068..4092
                     /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             join(4177..4617,1..276)
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFAYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(4207..4235)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
ORIGIN
        1 cacaatgtat acatcatggc agacaaacaa aagaatggaa tcaaagttaa cttcaaaatt
       61 agacacaaca ttgaagatgg aagcgttcaa ctagcagacc attatcaaca aaatactcca
      121 attggcgatg gccctgtcct tttaccagac aaccattacc tgtccacaca atctgccctt
      181 tcgaaagatc ccaacgaaaa gagagaccac atggtccttc ttgagtttgt aacagctgct
      241 gggattacac atggcatgga tgaactatac aaatagaatt agactagcat aaccccttgg
      301 ggcctctaaa cgggtcttga ggggtttttt gcactagcat aaccccttgg ggcctctaaa
      361 cgggtcttga ggggtttttt gcgaattcga tctcaattgg atattccact cgagtctggt
      421 aaagaaaccg ctgctgcgaa atttgaacgc cagcacatgg actcgtctac tagcgcagct
      481 taattaacct aggctgctgc caccgctgag caataactag cataacccct tggggcctct
      541 aaacgggtct tgaggggttt tttgctgaaa cctcaggcat ttgagaagca cacggtcaca
      601 ctgcttccgg tagtcaataa accggtaaac cagcaataga cataagcggc tatttaacga
      661 ccctgccctg aaccgacgac cgggtcgaat ttgctttcga atttctgcca ttcatccgct
      721 tattatcact tattcaggcg tagcaaccag gcgtttaagg gcaccaataa ctgccttaaa
      781 aaaattacgc cccgccctgc cactcatcgc agtactgttg taattcatta agcattctgc
      841 cgacatggaa gccatcacaa acggcatgat gaacctgaat cgccagcggc atcagcacct
      901 tgtcgccttg cgtataatat ttgcccatag tgaaaacggg ggcgaagaag ttgtccatat
      961 tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg attggctgag acgaaaaaca
     1021 tattctcaat aaacccttta gggaaatagg ccaggttttc accgtaacac gccacatctt
     1081 gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta ttcactccag agcgatgaaa
     1141 acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg aacactatcc catatcacca
     1201 gctcaccgtc tttcattgcc atacggaact ccggatgagc attcatcagg cgggcaagaa
     1261 tgtgaataaa ggccggataa aacttgtgct tatttttctt tacggtcttt aaaaaggccg
     1321 taatatccag ctgaacggtc tggttatagg tacattgagc aactgactga aatgcctcaa
     1381 aatgttcttt acgatgccat tgggatatat caacggtggt atatccagtg atttttttct
     1441 ccattttagc ttccttagct cctgaaaatc tcgataactc aaaaaatacg cccggtagtg
     1501 atcttatttc attatggtga aagttggaac ctcttacgtg ccgatcaacg tctcattttc
     1561 gccaaaagtt ggcccagggc ttcccggtat caacagggac accaggattt atttattctg
     1621 cgaagtgatc ttccgtcaca ggtatttatt cggcgcaaag tgcgtcgggt gatgctgcca
     1681 acttactgat ttagtgtatg atggtgtttt tgaggtgctc cagtggcttc tgtttctatc
     1741 agctgtccct cctgttcagc tactgacggg gtggtgcgta acggcaaaag caccgccgga
     1801 catcagcgct agcggagtgt atactggctt actatgttgg cactgatgag ggtgtcagtg
     1861 aagtgcttca tgtggcagga gaaaaaaggc tgcaccggtg cgtcagcaga atatgtgata
     1921 caggatatat tccgcttcct cgctcactga ctcgctacgc tcggtcgttc gactgcggcg
     1981 agcggaaatg gcttacgaac ggggcggaga tttcctggaa gatgccagga agatacttaa
     2041 cagggaagtg agagggccgc ggcaaagccg tttttccata ggctccgccc ccctgacaag
     2101 catcacgaaa tctgacgctc aaatcagtgg tggcgaaacc cgacaggact ataaagatac
     2161 caggcgtttc cccctggcgg ctccctcgtg cgctctcctg ttcctgcctt tcggtttacc
     2221 ggtgtcattc cgctgttatg gccgcgtttg tctcattcca cgcctgacac tcagttccgg
     2281 gtaggcagtt cgctccaagc tggactgtat gcacgaaccc cccgttcagt ccgaccgctg
     2341 cgccttatcc ggtaactatc gtcttgagtc caacccggaa agacatgcaa aagcaccact
     2401 ggcagcagcc actggtaatt gatttagagg agttagtctt gaagtcatgc gccggttaag
     2461 gctaaactga aaggacaagt tttggtgact gcgctcctcc aagccagtta cctcggttca
     2521 aagagttggt agctcagaga accttcgaaa aaccgccctg caaggcggtt ttttcgtttt
     2581 cagagcaaga gattacgcgc agaccaaaac gatctcaaga agatcatctt attaatcaga
     2641 taaaatattt ctagatttca gtgcaattta tctcttcaaa tgtagcacct gaagtcagcc
     2701 ccatacgata taagttgtaa ttctcatgtt agtcatgccc cgcgcccacc ggaaggagct
     2761 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     2821 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     2881 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     2941 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     3001 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     3061 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     3121 agatgtccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     3181 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     3241 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     3301 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     3361 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     3421 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     3481 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     3541 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     3601 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     3661 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     3721 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     3781 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     3841 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     3901 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     3961 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     4021 cccttatgcg actcctgcat taggaaatta atacgactca ctatagggga attgtgagcg
     4081 gataacaatt cccctgtaga aataattttg tttaacttta attctagagt cacacaggac
     4141 tactagatgg ggatgaccat gattacgcca agctttatga gtaaaggaga agaacttttc
     4201 actggagttg tcccaattct tgttgaatta gatggtgatg ttaatgggca caaattttct
     4261 gtcagtggag agggtgaagg tgatgcaaca tacggaaaac ttacccttaa atttatttgc
     4321 actactggaa aactacctgt tccatggcca acacttgtca ctactttcgc gtatggtctt
     4381 caatgctttg cgagataccc agatcatatg aaacagcatg actttttcaa gagtgccatg
     4441 cccgaaggtt atgtacagga aagaactata tttttcaaag atgacgggaa ctacaagaca
     4501 cgtgctgaag tcaaatttga aggtgatacc cttgttaata gaatcgagtt aaaaggtatt
     4561 gattttaaag aagatggaaa cattcttgga cacaaattgg aatacaacta taactca
//
