LOCUS       sequence-213340-        6089 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses gfp in mycobacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6089)
  AUTHORS   Eitson JL, Medeiros JJ, Hoover AR, Srivastava S, Roybal KT, Ainsa 
            JA, Hansen EJ, Gumbo T, van Oers NS
  TITLE     Mycobacterial shuttle vectors designed for high-level protein 
            expression in infected macrophages.
  JOURNAL   Appl Environ Microbiol. 2012 Oct;78(19):6829-37. doi: 
            10.1128/AEM.01674-12. Epub 2012 Jul 20.
  PUBMED    22820329
REFERENCE   2  (bases 1 to 6089)
  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: 
            "10.1128/AEM.01674-12"; journalName: "Appl Environ Microbiol"; date:
            "2012-10"; volume: "78"; issue: "19"; pages: "6829-37"
COMMENT     Sequence Label: pSum-LacZ-gfp
FEATURES             Location/Qualifiers
     source          1..6089
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(15..20,21..38,39..45)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    53..69
                     /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)."
     primer_bind     58..80
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     77..93
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     77..93
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             212..925
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(242..270)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     865..885
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     misc_feature    929..985
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(989..1006)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(989..1005)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(998..1020)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(1385..2179)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1496..1515)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2106..2125
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     rep_origin      5152..5697
                     /direction=RIGHT
                     /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     5598..5617
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5814..5831
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6068..6089
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
       61 ggataacaat ttcacacagg aaacagctat gaccatgatt acgaattccc ggggatccgt
      121 cgacctgcag ccaagcttgg gctgcaggtc gactctagag gatccccggg cgagctcgaa
      181 ttctgattaa ctttataagg aggaaaaaca tatgagtaaa ggagaagaac ttttcactgg
      241 agttgtccca attcttgttg aattagatgg tgatgttaat gggcacaaat tttctgtcag
      301 tggagagggt gaaggtgatg caacatacgg aaaacttacc cttaaattta tttgcactac
      361 tggaaaacta cctgttccat ggccaacact tgtcactact ttgacttatg gtgttcaatg
      421 cttttcaaga tacccagatc atatgaaacg gcatgacttt ttcaagagtg ccatgcccga
      481 aggttatgta caggaaagaa ctatattttt caaagatgac gggaactata agacacgtgc
      541 tgaagtcaag tttgaaggtg atacacttgt taatagaatc gagttaaaag gtattgattt
      601 taaagaagat ggaaacattc ttggacacaa attggaatac aactataact cacacaatgt
      661 atacatcatg gcagacaaac aaaagaatgg aatcaaagtt aacttcaaaa ttagacacaa
      721 cattgaagat ggaagcgttc aactagcaga ccattatcaa caaaatactc caattggcga
      781 tggccctgtc cttttaccag acaaccatta cctgtccaca caatctgccc tttcgaaaga
      841 tcccaacgaa aagagagacc acatggtcct tcttgagttt gtaacagctg ctgggattac
      901 acatggcatg gatgaactat acaaatagga attcgagctc ggtacccggg gatcctctag
      961 agtcgacctg caggcatgca agcttggcac tggccgtcgt tttacaacgt cgtgactggg
     1021 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     1081 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     1141 aatggcgctg atgtccggcg gtgcttttgc cgttacgcac caccccgtca gtagctgaac
     1201 aggagggaca gggggtgggc gaagaactcc agcatgagat ccccgcgctg gaggatcatc
     1261 cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc ggcggtggaa
     1321 tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg tcatttcgaa ccccagagtc
     1381 ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg
     1441 cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct tcagcaatat
     1501 cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg ccacagtcga
     1561 tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca tcgccatggg
     1621 tcacgacgag atcctcgccg tcgggcatcc gcgccttgag cctggcgaac agttcggctg
     1681 gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg gcttccatcc
     1741 gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag gtagccggat
     1801 caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg gcaggagcaa
     1861 ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag tcccttcccg
     1921 cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc agccacgata
     1981 gccgcgctgc ctcgtcttgg agttcattca gggcaccgga caggtcggtc ttgacaaaaa
     2041 gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag ccgattgtct
     2101 gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa cctgcgtgca
     2161 atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca gatcttgatc
     2221 ccctgcgcca tcagatcctt ggcggcaaga aagccatcca gtttactttg cagggcttcc
     2281 caaccttacc agagggcgcc ccagctggca attccggttc gcttgctgtc cataaaaccg
     2341 cccagtctag ctatcgccat gtaagcccac tgcaagctac ctgctttctc tttgcgcttg
     2401 cgttttccct tgtccagata gcccagtagc tgacattcat ccggggtcag caccgtttct
     2461 gcggactggc tttctacgtg ttccgcttcc tttagcagcc cttgcgccct gagtgcttgc
     2521 ggcagcgtga agctgtgcct agaaataacg aggacagtcg cacgacgaag ttcttctgga
     2581 tcgcgcccgt gctggaagca ctcaacctcg aagcgtgtgg ttgcggagcc atctagcaac
     2641 cacacgaaac atgcgcaacg aaccgcgcaa cgaacaacgc ctagaactgg ccctagatga
     2701 gctgactcgt atcgttggta aacctagttt gaccagcatg ttttaactac gttcggtgag
     2761 ctgtcaacgg ggcctgtaac ggcacaacga accgtgcaac gagagtggcc acggatgcca
     2821 ccacaggcac tacaacggag ttcgccacgt acatcaccac aaccaccgat tctggcggtg
     2881 agctccccga tattcagcgg aaatggcttg gtatcgacca agattcgtag aaccccgtct
     2941 cgtctggctg gtattcaaaa cgggcgcaac gaaacacgca acgagacagg catggcccaa
     3001 accagaaaac tagcgtctac caggactttt acctgtccga cccgttgcaa cggaaccccc
     3061 cacggaaccc ccgcgacacc cgctccccaa ttgcgttaga acagcggtgg attgtcggct
     3121 tcgttgtggg ccttttgagc cgcttcctgt tctgccgcac gctctttcct cgcccgatag
     3181 ccgagtcgct taacggtgtc cagatgcagc ccgaaatgtt tggccgtttg cggccaagag
     3241 tggccctcgt cgtcgtgata ggcgcggatg cgttcgcggc gtgcagcctg ctcggcgagc
     3301 cactcgctgc gttcctgcgc cacgagccgg acgacgtggc gttcggatag tccggtgatt
     3361 cgagcgcctt cggcggcggt cacgcgccgc tttttgcgga cagtcggctg ccggttgtag
     3421 ccgtcgctgt agccgtcgct catagcaatg cctccatggc tgacgcggac tttgcgcgcc
     3481 gcgcaactgt gctcgccgcc gtgcgcgctg ctgcgccctt ccgcgagatg gccgactggc
     3541 gcgcactgag tgtggcctcg tagaccacga tcccgtccgc ccaaatgcgc gacttggttg
     3601 tgatccaacg ccaaatgctg ttggcgatgg cgcggacctc gctgtccggt agcggtccgg
     3661 gacacacgtc gttgcacggg aattcggcgt ttcgcgcgtg gcactcggca tagatcgcgc
     3721 ggccgagtcc gtccacgttc cgggtcggca ggtagatccg catgagggcg ggacgatagg
     3781 cccacaacct gacggaatcg aacagtgcgc aattccgccc tagcggcgtc ggagccgctt
     3841 tgtacgtggt ctgctgacgc cagcgcggcg gtggcatgtt cgcgccgagc tcggcctcga
     3901 tgtggctgag tgtgtagaga tctgagtgga gccattccgt ttcccaggcg atgtggccgg
     3961 ggtttttggt catgaggcct gagtaactgc ggtcgccgtc gacggcgcgc cgaaggcctt
     4021 cggcgcacgc cgccatgtat gcgagcggct tacgccgcgc gtattcggtg cgtggaacag
     4081 gggcgttgag tgcccacact gcgtgtgcgt ggccgttggc gcgattgccc acgatcgcgt
     4141 tgggcagcgg atgggacccc cgggcgctga gcgctcggag cgctgcgtct ggatggtcta
     4201 cgtccacgac cagcaggttt gccagcgctg ttgggttcgc ctcgatgtac cggcggccta
     4261 gggccgacgc gcggctttgg cggtagatcc cctcgagcag atcgtcgctt gccagcggcc
     4321 agtacggcag ccagagctgc tcaaattcgt cggcgacgtg gctcacgctt ggtagtagac
     4381 cacgattaat caccggtgta tggtccgaca cgagctccaa gtcagatatt tcgctgaggg
     4441 gccaccccac aactgcacac tcccccgctc tcccgtcgag ccctggtggt ggaacaccag
     4501 cgacagccga gcacccccaa ccacctgtac caaccaggag gaacacatgc gtcgtttcga
     4561 ggacgtttcc gggccgctga gagccgctgt ggcggccgta cacgccgcct tagacccgtt
     4621 agaccccctg ccgcctgaat gcgcgggtac gagccacaca gcgcccgaac ttacggagct
     4681 ggtgggctca cctggcttta tggcgtacga atcggctgtg tgcgacctgt tgggcgaggt
     4741 gaggtacgcg ctactcacgc tggcaagggc gacagagccg ccccaccgag cccgcacggc
     4801 cgcgcgcggt gtcaacaacc gggtgagtcg tgcacaccag caggtgttcg aggcttggct
     4861 cgaagtgcag gacatcgtgg cgaacgccgc ccgatgagcc gcgccttacg ctggctgcca
     4921 gccgttcgcg ggctggttgg tgcagcgcgt cgagcggtta gaggccctgc ggtgttccac
     4981 caccgcaggg cctcgccctt tttaaggctg aatttgcttg tctccgaatc caactggctt
     5041 gtccaagggt gtatctacgc ttaatccaaa gttcaaacga ggggattaca catgaccaac
     5101 ttcgataacg ttctcggctc gatattttat ctgattaata agatgatctt cttgagatcg
     5161 ttttggtctg cgcgtaatct cttgctctga aaacgaaaaa accgccttgc agggcggttt
     5221 ttcgaaggtt ctctgagcta ccaactcttt gaaccgaggt aactggcttg gaggagcgca
     5281 gtcaccaaaa cttgtccttt cagtttagcc ttaaccggcg catgacttca agactaactc
     5341 ctctaaatca attaccagtg gctgctgcca gtggtgcttt tgcatgtctt tccgggttgg
     5401 actcaagacg atagttaccg gataaggcgc agcggtcgga ctgaacgggg ggttcgtgca
     5461 tacagtccag cttggagcga actgcctacc cggaactgag tgtcaggcgt ggaatgagac
     5521 aaacgcggcc ataacagcgg aatgacaccg gtaaaccgaa aggcaggaac aggagagcgc
     5581 acgagggagc cgccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     5641 cactgatttg agcgtcagat ttcgtgatgc ttgtcagggg ggcggagcct atggaaaaac
     5701 ggctttgccg cggccctctc acttccctgt taagtatctt cctggcatct tccaggaaat
     5761 ctccgccccg ttcgtaagcc atttccgctc gccgcagtcg aacgaccgag cgtagcgagt
     5821 cagtgagcga ggaagcggaa tatatcctgt atcacatatt ctgctgacgc accggtgcag
     5881 ccttttttct cctgccacat gaagcacttc actgacaccc tcatcagtgc caacatagta
     5941 agccagtata tacactccgc tagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     6001 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     6061 acgcaattaa tgtgagttag ctcactcat
//
