LOCUS       sequence-378574-        5952 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a GFP design, mid.2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5952)
  AUTHORS   Weinstein JY, Marti-Gomez C, Lipsh-Sokolik R, Hoch SY, Liebermann D,
            Nevo R, Weissman H, Petrovich-Kopitman E, Margulies D, Ivankov D, 
            McCandlish DM, Fleishman SJ
  TITLE     Designed active-site library reveals thousands of functional GFP 
            variants.
  JOURNAL   Nat Commun. 2023 May 20;14(1):2890. doi: 10.1038/s41467-023-38099-z.
  PUBMED    37210560
REFERENCE   2  (bases 1 to 5952)
  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: "Nat 
            Commun."; date: "2023-05-20"; volume: "14(1):2890. doi"; pages: " 
            10.1038/s41467-023-38099-z"
COMMENT     Sequence Label: pET28 mid.2
FEATURES             Location/Qualifiers
     source          1..5952
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(887..905)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      901..948
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      985..1440
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1072..1091)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1282..1303
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1532..2347)
                     /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             complement(1532..2347)
                     /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     2255..2274
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2339..2358
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2469..3057
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2958..2977
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3211..3228
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3243..3382
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3308..3327)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3468..3490
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3484..3675)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4484..5566)
                     /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     5528..5547
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5567..5644)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5850..5869
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacgtt
      121 agcaagggcg aggagctgtt caccggcgtt gttccaatct tagtagagtt agacggagat
      181 gtaaatggcc acaagttttc ggttcgaggc gagggagaag gagacgcgac gtatggcaaa
      241 ctgacgctga aattcatttg tacgaccgga aaactgcctg tgccgtggcc tacccttgta
      301 acaaccctta cgtatggtgt acaatgcttt tcccgttatc ctgatcacat gaaacagcac
      361 gattttttca aatctgcaat gcctgagggt tacgtacagg aacgcaccat tttttttaag
      421 gatgacggtt gttacaagat tcgcgcagaa gttaagtttg agggcgatac cttagtcaat
      481 cgtattgagc tgcatggtat tgactttaaa gaggacggca acattttggg acacaaactt
      541 gagtacaata ttaatagtca caacgtgtat ataatgccag acaaacaaaa caacggaatt
      601 aaagtcaact tcaaaaccag acataacatc gaagacggca gcgttcagct cgctgatcat
      661 tatcagcaaa ataccccgat tggtgatggg ccagtgcttc ttccagacta ccattatctc
      721 catacatggt cagaactctc caaagatccg aatgaaaaac gagatcacat ggttctgctg
      781 gaatttgtag aagcggctgg tattactctc ggcatggacg agctgtataa gtaatgagat
      841 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
      901 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
      961 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     1021 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     1081 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     1141 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     1201 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     1261 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     1321 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     1381 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     1441 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     1501 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     1561 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     1621 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     1681 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     1741 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     1801 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     1861 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     1921 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     1981 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     2041 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     2101 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     2161 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     2221 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     2281 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     2341 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     2401 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     2461 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     2521 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     2581 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     2641 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     2701 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     2761 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     2821 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     2881 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     2941 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     3001 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     3061 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     3121 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     3181 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     3241 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     3301 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     3361 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     3421 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     3481 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     3541 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     3601 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     3661 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     3721 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     3781 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     3841 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     3901 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     3961 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     4021 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     4081 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     4141 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     4201 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     4261 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     4321 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     4381 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     4441 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     4501 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4561 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     4621 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     4681 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     4741 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
     4801 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     4861 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     4921 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     4981 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     5041 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     5101 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     5161 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     5221 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     5281 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     5341 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     5401 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     5461 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     5521 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     5581 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     5641 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     5701 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
     5761 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
     5821 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
     5881 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg
     5941 atcccgcgaa at
//
