LOCUS       sequence-492406-        7013 bp    DNA     circular SYN 10-NOV-2025
DEFINITION  sfGFP constitutive Golden Gate construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 4118 to 4152)
  TITLE     Murray Lab Golden Gate Plasmids 2025
REFERENCE   2  (bases 1 to 7013)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 10, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: eEML021
FEATURES             Location/Qualifiers
     source          1..7013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     mobile_element  1210..1375
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     primer_bind     complement(1494..1513)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1505..2320
                     /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             1505..2320
                     /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     complement(1578..1597)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4033..4051
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4118..4152
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     RBS             4161..4172
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             4179..4892
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      4903..4997
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      5402..5790
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     CDS             complement(5961..6332)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            6365..6474
                     /note="incP origin of transfer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc tgaattaatt cgagctcgcc caaacataac aggaagaaaa
     1201 atgccccgct gtgggcggac aaaatagttg ggaactggga ggggtggaaa tggagttttt
     1261 aaggattatt tagggaagag tgacaaaata gatgggaact gggtgtagcg tcgtaagcta
     1321 atacgaaaat taaaaatgac aaaatagttt ggaactagat ttcacttatc tggttggcct
     1381 gcaggggggg ggggaaagcc acgttgtgtc tcaaaatctc tgatgttaca ttgcacaaga
     1441 taaaaatata tcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg
     1501 tgttatgagc catattcaac gggaaacgtc ttgctcgagg ccgcgattaa attccaacat
     1561 ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac
     1621 aatctatcga ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg
     1681 tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat
     1741 gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac
     1801 tgcgatcccc gggaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa
     1861 tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg
     1921 tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg
     1981 tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg
     2041 gaaagaaatg cataagcttt tgccattctc accggattca gtcgtcactc atggtgattt
     2101 ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg
     2161 agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt
     2221 ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa
     2281 taaattgcag tttcatttga tgctcgatga gtttttctaa tcagaattgg ttaattggtt
     2341 gtaacactgg cagagcatta cgctgacttg acgggacggc ggctttgttg aataaatcga
     2401 acttttgctg agttgaagga tcagatcacg catcttcccg acaacgcaga ccgttccgtg
     2461 gcaaagcaaa agttcaaaat caccaactgg tccacctaca acaaagctct catcaaccgt
     2521 ggctccctca ctttctggct ggatgatggg gcgattcagg cctggtatga gtcagcaaca
     2581 ccttcttcac gaggcagacc tcagcgcccc ccccccctgc aggtcgacta gagtcgggga
     2641 tccggtgatt gattgagcaa gctttatgct tgtaaaccgt tttgtgaaaa aatttttaaa
     2701 ataaaaaagg ggacctctag ggtccccaat taattagtaa tataatctat taaaggtcat
     2761 tcaaaaggtc atccaccgga tcaattcccc tgctcgcgca ggctgggtgc caagctctcg
     2821 ggtaacatca aggcccgatc cttggagccc ttgccctccc gcacgatgat cgtgccgtga
     2881 tcgaaatcca gatccttgac ccgcagttgc aaaccctcac tgatccgcat gcccgttcca
     2941 tacagaagct gggcgaacaa acgatgctcg ccttccagaa aaccgaggat gcgaaccact
     3001 tcatccgggg tcagcaccac cggcaagcgc cgcgacggcc gaggtcttcc gatctcctga
     3061 agccagggca gatccgtgca cagcaccttg ccgtagaaga acagcaaggc cgccaatgcc
     3121 tgacgatgcg tggagaccga aaccttgcgc tcgttcgcca gccaggacag aaatgcctcg
     3181 acttcgctgc tgcccaaggt tgccgggtga cgcacaccgt ggaaacggat gaaggcacga
     3241 acccagtgga cataagcctg ttcggttcgt aagctgtaat gcaagtagcg tatgcgctca
     3301 cgcaactggt ccagaacctt gaccgaacgc agcggtggta acggcgcagt ggcggttttc
     3361 atggcttgtt atgactgttt ttttggggta cagtctatgc ctcgggcatc caagcagcaa
     3421 gcgcgttacg ccgtgggtcg atgtttgtgt ctaacaattc gttcaagccg acgccgcttc
     3481 gcggcgcggc ttaactcaag cgttagatgc actaagcaca taattgctca cagccaaact
     3541 atcaggtcaa gtctgctttt attattttta agcgtgcata ataagcccta cacaaattgg
     3601 gagatatatc atgaaaggct ggctttttct tgttatcgca atagttggcg aagtaatcgc
     3661 aacatccgca ttaaaatcta gcgagggctt tactaagctg atccggtgga tgaccttttg
     3721 aatgaccttt aatagattat attactaatt aattggggac cctagaggtc ccctttttta
     3781 ttttaaaaat tttttcacaa aacggtttac aagcataaag cttgctcaat caatcaccgg
     3841 atcccgacgg gcccggtacc gcggccgcgc gaattcgagc tcggtaccat ttatcagggt
     3901 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     3961 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     4021 ttaacctata aaaataggcg tatcacgagg ccctttcgtc ttcacctcga gaacattact
     4081 cgcatccatt ctcaggctgt ctcgtctcgt ctcggagttg acggctagct cagtcctagg
     4141 tacagtgcta gctactagag aaagaggaga aatactaaat gcgtaaaggc gaagagctgt
     4201 tcactggtgt cgtccctatt ctggtggaac tggatggtga tgtcaacggt cataagtttt
     4261 ccgtgcgtgg cgagggtgaa ggtgacgcaa ctaatggtaa actgacgctg aagttcatct
     4321 gtactactgg taaactgccg gtaccttggc cgactctggt aacgacgctg acttatggtg
     4381 ttcagtgctt tgctcgttat ccggaccata tgaagcagca tgacttcttc aagtccgcca
     4441 tgccggaagg ctatgtgcag gaacgcacga tttcctttaa ggatgacggc acgtacaaaa
     4501 cgcgtgcgga agtgaaattt gaaggcgata ccctggtaaa ccgcattgag ctgaaaggca
     4561 ttgactttaa agaagatggc aatatcctgg gccataagct ggaatacaat tttaacagcc
     4621 acaatgttta catcaccgcc gataaacaaa aaaatggcat taaagcgaat tttaaaattc
     4681 gccacaacgt ggaggatggc agcgtgcagc tggctgatca ctaccagcaa aacactccaa
     4741 tcggtgatgg tcctgttctg ctgccagaca atcactatct gagcacgcaa agcgttctgt
     4801 ctaaagatcc gaacgagaaa cgcgatcata tggttctgct ggagttcgta accgcagcgg
     4861 gcatcacgca tggtatggat gaactgtaca aatgatgaag gtgactcctg ttgatagatc
     4921 cagtaatgac ctcagaactc catctggatt tgttcagaac gctcggttgc cgccgggcgt
     4981 tttttattgg tgagaatgct tccaggatac atagattacc acaactccga gcccttccac
     5041 cccaagctag cttggaggac aaatccgccg ctaggagctt gcggccgcct taagacgcgt
     5101 ggatccccgg gaatttcgac cccacgcccc tctttaatac gacgggcaat ttgcacttca
     5161 gaaaatgaag agtttgcttt agccataaca aaagtccagt atgctttttc acagcataac
     5221 tggactgatt tcagtttaca actattctgt ctagtttaag actttattgt catagtttag
     5281 atctattttg ttcagtttaa gactttattg tccgcccaca cccgcttacg cagggcatcc
     5341 atttattact caaccgtaac cgattttgcc aggttacgcg gctggtcctc tagaattccc
     5401 atgtcagccg ttaagtgttc ctgtgtcact caaaattgct ttgagaggct ctaagggctt
     5461 ctcagtgcgt tacatccctg gcttgttgtc cacaaccgtt aaaccttaaa agctttaaaa
     5521 gccttatata ttcttttttt tcttataaaa cttaaaacct tagaggctat ttaagttgct
     5581 gatttatatt aattttattg ttcaaacatg agagcttagt acgtgaaaca tgagagctta
     5641 gtacgttagc catgagagct tagtacgtta gccatgaggg tttagttcgt taaacatgag
     5701 agcttagtac gttaaacatg agagcttagt acgtgaaaca tgagagctta gtacgtacta
     5761 tcaacaggtt gaactgctga tcttcagatc ctctacgccg gacgcatcgt ggccggatcg
     5821 gggggggggg gcgctcggtc ttgccttgct cgtcggtgat gtacttcacc agctccgcga
     5881 agtcgctctt cttgatggag cgcatgggga cgtgcttggc aatcacgcgc accccccggc
     5941 cgttttagcg gctaaaaaag tcatggctct gccctcgggc ggaccacgcc catcatgacc
     6001 ttgccaagct cgtcctgctt ctcttcgatc ttcgccagca gggcgaggat cgtggcatca
     6061 ccgaaccgcg ccgtgcgcgg gtcgtcggtg agccagagtt tcagcaggcc gcccaggcgg
     6121 cccaggtcgc cattgatgcg ggccagctcg cggacgtgct catagtccac gacgcccgtg
     6181 attttgtagc cctggccgac ggccagcagg taggccgaca ggctcatgcc ggccgccgcc
     6241 gccttttcct caatcgctct tcgttcgtct ggaaggcagt acaccttgat aggtgggctg
     6301 cccttcctgg ttggcttggt ttcatcagcc atccgcttgc cctcatctgt tacgccggcg
     6361 gtagccggcc agcctcgcag agcaggattc ccgttgagca ccgccaggtg cgaataaggg
     6421 acagtgaaga aggaacaccc gctcgcgggt gggcctactt cacctatcct gcccggctga
     6481 cgccgttgga tacaccaagg aaagtctaca cgaacccttt ggcaaaatcc tgtatatcgt
     6541 gcgaaaaagg atggatatac cgaaaaaatc gctataatga ccccgaagca gggttatgca
     6601 gcggaaaagc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     6661 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     6721 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     6781 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     6841 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     6901 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     6961 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caa
//
