LOCUS       sequence-373764-        6152 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An E. coli sfGFP reporter with two TAG at positions 2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6152)
  AUTHORS   Wu KL, Moore JA, Miller MD, Chen Y, Lee C, Xu W, Peng Z, Duan Q, 
            Phillips GN Jr, Uribe RA, Xiao H
  TITLE     Expanding the eukaryotic genetic code with a biosynthesized 21st 
            amino acid.
  JOURNAL   Protein Sci. 2022 Oct;31(10):e4443. doi: 10.1002/pro.4443.
  PUBMED    36173166
REFERENCE   2  (bases 1 to 6152)
  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"; journalName: "Protein 
            Sci."; date: "2022-10"; volume: "31(10):e4443. doi"; pages: " 
            10.1002/pro.4443"
COMMENT     Sequence Label: pLei-sfGFP-V2*D134*Y152*
FEATURES             Location/Qualifiers
     source          1..6152
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(220..322)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(714..731)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(848..1393)
                     /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(928..947)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(1534..1548,1549..1554,1555..1571,1572..1578)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    1554..1570
                     /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)."
     protein_bind    1586..1602
                     /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)."
     RBS             1621..1632
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1639..2355
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="M*SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKE*GNILGHKLEYNFNSHNV*ITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(1672..1700)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2295..2315
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             2362..2379
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2402..2496
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             2540..3199
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     terminator      3264..3350
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        3486..3563
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3564..4646
                     /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
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSCADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQAPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(3583..3602)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     primer_bind     4830..4852
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4844..4861
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4845..4861
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(join(5712..6152,1..219))
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVLHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFATVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
ORIGIN
        1 gaactccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt
       61 gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt
      121 ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga
      181 tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga
      241 aaatctcgat aactcaaaaa atacgcccgg tagtgatctt atttcattat ggtgaaagtt
      301 ggaacctctt acgtgccgat caacgtctca ttttcgccaa aagttggccc agggcttccc
      361 ggtatcaaca gggacaccag gatttattta ttctgcgaag tgatcttccg tcacaggtat
      421 ttattcggcg caaagtgcgt cgggtgatgc tgccaactta ctgatttagt gtatgatggt
      481 gtttttgagg tgctccagtg gcttctgttt ctatcagctg tccctcctgt tcagctactg
      541 acggggtggt gcgtaacggc aaaagcaccg ccggacatca gcgctagcgg agtgtatact
      601 ggcttactat gttggcactg atgagggtgt cagtgaagtg cttcatgtgg caggagaaaa
      661 aaggctgcac cggtgcgtca gcagaatatg tgatacagga tatattccgc ttcctcgctc
      721 actgactcgc tacgctcggt cgttcgactg cggcgagcgg aaatggctta cgaacggggc
      781 ggagatttcc tggaagatgc caggaagata cttaacaggg aagtgagagg gccgcggcaa
      841 agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga cgctcaaatc
      901 agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggcggctccc
      961 tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt cattccgctg ttatggccgc
     1021 gtttgtctca ttccacgcct gacactcagt tccgggtagg cagttcgctc caagctggac
     1081 tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt
     1141 gagtccaacc cggaaagaca tgcaaaagca ccactggcag cagccactgg taattgattt
     1201 agaggagtta gtcttgaagt catgcgccgg ttaaggctaa actgaaagga caagttttgg
     1261 tgactgcgct cctccaagcc agttacctcg gttcaaagag ttggtagctc agagaacctt
     1321 cgaaaaaccg ccctgcaagg cggttttttc gttttcagag caagagatta cgcgcagacc
     1381 aaaacgatct caagaagatc atcttattaa tcagataaaa tatttctaga tttcagtgca
     1441 atttatctct tcaaatgtag cacctgaagt cagccccata cgatataagt tgtaattctc
     1501 atgtttgaca gcattatcat cgatctcgag aaatcataaa aaatttattt gctttgtgag
     1561 cggataacaa ttataataga ttcaattgtg agcggataac aatttcacac agaattcatt
     1621 aaagaggaga aattacatat gtagagcaaa ggagaagaac ttttcactgg agttgtccca
     1681 attcttgttg aattagatgg tgatgttaat gggcacaaat tttctgtccg tggagagggt
     1741 gaaggtgatg ctacaaacgg aaaactcacc cttaaattta tttgcactac tggaaaacta
     1801 cctgttccgt ggccaacact tgtcactact ctgacctatg gtgttcaatg cttttcccgt
     1861 tatccggatc acatgaaacg gcatgacttt ttcaagagtg ccatgcccga aggttatgta
     1921 caggaacgca ctatatcttt caaagatgac gggacctaca agacgcgtgc tgaagtcaag
     1981 tttgaaggtg atacccttgt taatcgtatc gagttaaagg gtattgattt taaagaatag
     2041 ggaaacattc ttggacacaa actcgagtac aactttaact cacacaatgt atagatcacg
     2101 gcagacaaac aaaagaatgg aatcaaagct aacttcaaaa ttcgccacaa cgttgaagat
     2161 ggttccgttc aactagcaga ccattatcaa caaaatactc caattggcga tggccctgtc
     2221 cttttaccag acaaccatta cctgtcgaca caatctgccc tttcgaaaga tcccaacgaa
     2281 aagcgtgacc acatggtcct tcttgagttt gtaactgctg ctgggattac acatggcatg
     2341 gatgaactct acaaagagct ccatcaccat caccatcact aagcttaatt agctgagctt
     2401 ggactcctgt tgatagatcc agtaatgacc tcagaactcc atctggattt gttcagaacg
     2461 ctcggttgcc gccgggcgtt ttttattggt gagaatccaa gctagcttgg cgagattttc
     2521 aggagctaag gaagctaaaa tggagaaaaa aatcactgga tataccaccg ttgatatatc
     2581 ccaatggcat cgtaaagaac attttgaggc atttcagtca gttgctcaat gtacctataa
     2641 ccagaccgtt cagctggata ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa
     2701 gttttatccg gcctttattc acattcttgc ccgcctgatg aatgctcatc cggaatttcg
     2761 tatggcaatg aaagacggtg agctggtgat atgggatagt gttcaccctt gttacaccgt
     2821 tttccatgag caaactgaaa cgttttcatc gctctggagt gaataccacg acgatttccg
     2881 gcagtttcta cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt
     2941 ccctaaaggg tttattgaga atatgttttt cgtctcagcc aatccctggg tgagtttcac
     3001 cagttttgat ttaaacgtgg ccaatatgga caacttcttc gcccccgttt tcaccatggg
     3061 caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg ttcatcatgc
     3121 cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt actgcgatga
     3181 gtggcagggc ggggcgtaat ttttttaagg cagttattgg tgcccttaaa cgcctggggt
     3241 aatgactctc tagcttgagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct
     3301 ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccctcta
     3361 gatgcgcacc cgttctcgga gcactgtccg accgctttgg ccgccgccca gtcctgctcg
     3421 cttcgctact tggagccact atcgactacg cgatcatggc gaccacaccc gtcctgtgga
     3481 tccttgacac catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag
     3541 agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg
     3601 ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtctctgcga
     3661 aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg
     3721 cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc
     3781 tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatcttg cgccgatcaa ctgggtgcca
     3841 gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca
     3901 atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg
     3961 ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc
     4021 aggcacccat caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc
     4081 atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct
     4141 cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga
     4201 tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc
     4261 tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg
     4321 caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat
     4381 acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt
     4441 ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg
     4501 tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca
     4561 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     4621 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gcgcgaattg
     4681 tcggcatgca ccattccttg cggcggcggt gctcaacggc ctcaacctac tactgggctg
     4741 cttcctaatg caggagtcgc ataagggaga gcgtctggcg aaagggggat gtgctgcaag
     4801 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag
     4861 tgccaagctt aaaaaaaatc cttagctttc gctaaggatc tgcagtggcg gaaaccccgg
     4921 gaatctaacc cggctgaacg gatttagagt ccattcgatc tacatgatca ggtttccgag
     4981 ctcagcgtta caagtattac acaaagtttt ttatgttgag aatatttttt tgatggggcg
     5041 ccacttattt ttgatcgttc gctcaaagaa gcggcgccag ggttgttttt cttttcacca
     5101 gactagtgag acgggcaaca gaacgccatg agcggcctca tttcttattc tgagttacaa
     5161 cagtccgcac cgctgccggt agctccttcc ggtgggcgcg gggcatgact atcgtcgccg
     5221 cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgcccaaca
     5281 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgccc tgcaccatta
     5341 tgttccggat ctgcatcgca ggatgctgct ggctaccctg tggaacacct acatctgtat
     5401 taacgaagcg ctaaccgttt ttatcatgct ctgggaggca gaataaatga tcatatcgtc
     5461 aattattacc tccacgggga gagcctgagc aaactggcct caggcatttg agaagcacac
     5521 ggtcacactg cttccggtag tcaataaacc ggtaaaccag caatagacat aagcggctat
     5581 ttaacgaccc tgccctgaac cgacgaccgg gtcgaatttg ctttcgaatt tctgccattc
     5641 atccgcttat tatcacttat tcaggcgtag caaccaggcg tttaagggca ccaataactg
     5701 ccttaaaaaa attacgcccc gccctgccac tcatcgcagt actgttgtaa ttcattaagc
     5761 attctgccga catggaagcc atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc
     5821 agcaccttgt cgccttgcgt ataatatttg cccatggtga aaacggtggc gaagaagttg
     5881 tccatattgg ccacgtttaa atcaaaactg gtgaaactca cccagggatt ggctgagacg
     5941 aaaaacatat tctcaataaa ccctttaggg aaataggcca ggttttcacc gtaacacgcc
     6001 acatcttgcg aatatatgtg tagaaactgc cggaaatcgt cgtggtattc actccagagc
     6061 gatgaaaacg tttcagtttg ctcatgtaaa acggtgtaac aagggtgaac actatcccat
     6121 atcaccagct caccgtcttt cattgccata cg
//
