LOCUS       sequence-199526-        4617 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP with a premature stop codon.
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.GFP1Amb
FEATURES             Location/Qualifiers
     source          1..4617
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      18..65
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      68..115
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             155..199
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(237..255)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      251..298
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(519..1178)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1093..1112
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1179..1281)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(1673..1690)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1807..2352)
                     /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(1887..1906)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2577..3659)
                     /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     3621..3640
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3660..3737)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3783..3802
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3783..3801
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3802..3826
                     /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(3911..4617,1..10)
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDAT*GKLTLK
                     FICTTGKLPVPWPTLVTTFAYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(3941..3969)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4564..4584
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
ORIGIN
        1 atacaaatag aattagacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
       61 ttttgcacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgcgaat
      121 tcgatctcaa ttggatattc cactcgagtc tggtaaagaa accgctgctg cgaaatttga
      181 acgccagcac atggactcgt ctactagcgc agcttaatta acctaggctg ctgccaccgc
      241 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
      301 gaaacctcag gcatttgaga agcacacggt cacactgctt ccggtagtca ataaaccggt
      361 aaaccagcaa tagacataag cggctattta acgaccctgc cctgaaccga cgaccgggtc
      421 gaatttgctt tcgaatttct gccattcatc cgcttattat cacttattca ggcgtagcaa
      481 ccaggcgttt aagggcacca ataactgcct taaaaaaatt acgccccgcc ctgccactca
      541 tcgcagtact gttgtaattc attaagcatt ctgccgacat ggaagccatc acaaacggca
      601 tgatgaacct gaatcgccag cggcatcagc accttgtcgc cttgcgtata atatttgccc
      661 atagtgaaaa cgggggcgaa gaagttgtcc atattggcca cgtttaaatc aaaactggtg
      721 aaactcaccc agggattggc tgagacgaaa aacatattct caataaaccc tttagggaaa
      781 taggccaggt tttcaccgta acacgccaca tcttgcgaat atatgtgtag aaactgccgg
      841 aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt cagtttgctc atggaaaacg
      901 gtgtaacaag ggtgaacact atcccatatc accagctcac cgtctttcat tgccatacgg
      961 aactccggat gagcattcat caggcgggca agaatgtgaa taaaggccgg ataaaacttg
     1021 tgcttatttt tctttacggt ctttaaaaag gccgtaatat ccagctgaac ggtctggtta
     1081 taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat
     1141 atatcaacgg tggtatatcc agtgattttt ttctccattt tagcttcctt agctcctgaa
     1201 aatctcgata actcaaaaaa tacgcccggt agtgatctta tttcattatg gtgaaagttg
     1261 gaacctctta cgtgccgatc aacgtctcat tttcgccaaa agttggccca gggcttcccg
     1321 gtatcaacag ggacaccagg atttatttat tctgcgaagt gatcttccgt cacaggtatt
     1381 tattcggcgc aaagtgcgtc gggtgatgct gccaacttac tgatttagtg tatgatggtg
     1441 tttttgaggt gctccagtgg cttctgtttc tatcagctgt ccctcctgtt cagctactga
     1501 cggggtggtg cgtaacggca aaagcaccgc cggacatcag cgctagcgga gtgtatactg
     1561 gcttactatg ttggcactga tgagggtgtc agtgaagtgc ttcatgtggc aggagaaaaa
     1621 aggctgcacc ggtgcgtcag cagaatatgt gatacaggat atattccgct tcctcgctca
     1681 ctgactcgct acgctcggtc gttcgactgc ggcgagcgga aatggcttac gaacggggcg
     1741 gagatttcct ggaagatgcc aggaagatac ttaacaggga agtgagaggg ccgcggcaaa
     1801 gccgtttttc cataggctcc gcccccctga caagcatcac gaaatctgac gctcaaatca
     1861 gtggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gcggctccct
     1921 cgtgcgctct cctgttcctg cctttcggtt taccggtgtc attccgctgt tatggccgcg
     1981 tttgtctcat tccacgcctg acactcagtt ccgggtaggc agttcgctcc aagctggact
     2041 gtatgcacga accccccgtt cagtccgacc gctgcgcctt atccggtaac tatcgtcttg
     2101 agtccaaccc ggaaagacat gcaaaagcac cactggcagc agccactggt aattgattta
     2161 gaggagttag tcttgaagtc atgcgccggt taaggctaaa ctgaaaggac aagttttggt
     2221 gactgcgctc ctccaagcca gttacctcgg ttcaaagagt tggtagctca gagaaccttc
     2281 gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc aagagattac gcgcagacca
     2341 aaacgatctc aagaagatca tcttattaat cagataaaat atttctagat ttcagtgcaa
     2401 tttatctctt caaatgtagc acctgaagtc agccccatac gatataagtt gtaattctca
     2461 tgttagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     2521 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     2581 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     2641 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     2701 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     2761 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     2821 gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg
     2881 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     2941 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     3001 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     3061 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     3121 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     3181 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     3241 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     3301 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     3361 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     3421 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     3481 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     3541 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     3601 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     3661 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     3721 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     3781 attaatacga ctcactatag gggaattgtg agcggataac aattcccctg tagaaataat
     3841 tttgtttaac tttaattcta gagtcacaca ggactactag atggggatga ccatgattac
     3901 gccaagcttt atgagtaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga
     3961 attagatggt gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc
     4021 aacataggga aaacttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg
     4081 gccaacactt gtcactactt tcgcgtatgg tcttcaatgc tttgcgagat acccagatca
     4141 tatgaaacag catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaaagaac
     4201 tatatttttc aaagatgacg ggaactacaa gacacgtgct gaagtcaaat ttgaaggtga
     4261 tacccttgtt aatagaatcg agttaaaagg tattgatttt aaagaagatg gaaacattct
     4321 tggacacaaa ttggaataca actataactc acacaatgta tacatcatgg cagacaaaca
     4381 aaagaatgga atcaaagtta acttcaaaat tagacacaac attgaagatg gaagcgttca
     4441 actagcagac cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga
     4501 caaccattac ctgtccacac aatctgccct ttcgaaagat cccaacgaaa agagagacca
     4561 catggtcctt cttgagtttg taacagctgc tgggattaca catggcatgg atgaact
//
