LOCUS       sequence-211597-        6003 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses high levels of gfp in mycobacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6003)
  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 6003)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-Kan-MCS2-gfp
FEATURES             Location/Qualifiers
     source          1..6003
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             66..776
                     /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="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(93..121)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     716..736
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(810..826)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(810..826)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(823..845)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    834..850
                     /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)."
     promoter        complement(join(858..864,865..882,883..888))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    903..924
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1161..1178)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1295..1840)
                     /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(1375..1394)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             4813..5607
                     /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(4867..4886)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5477..5496
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5972..5994
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5986..6003
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5987..6003
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gccaagctgc gagctcgaat tctgattaac tttataagga ggaaaaacat atgacagaag
       61 ctttcagtaa aggagaagaa cttttcactg gagttgtccc aattcttgtt gaattagatg
      121 gtgatgttaa tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat gcaacatacg
      181 gaaaacttac ccttaaattt atttgcacta ctggaaaact acctgttcca tggccaacac
      241 ttgtcactac tttgacttat ggtgttcaat gcttttcaag atacccagat catatgaaac
      301 ggcatgactt tttcaagagt gccatgcccg aaggttatgt acaggaaaga actatatttt
      361 tcaaagatga cgggaactat aagacacgtg ctgaagtcaa gtttgaaggt gatacacttg
      421 ttaatagaat cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt cttggacaca
      481 aattggaata caactataac tcacacaatg tatacatcat ggcagacaaa caaaagaatg
      541 gaatcaaagt taacttcaaa attagacaca acattgaaga tggaagcgtt caactagcag
      601 accattatca acaaaatact ccaattggcg atggccctgt ccttttacca gacaaccatt
      661 acctgtccac acaatctgcc ctttcgaaag atcccaacga aaagagagac cacatggtcc
      721 ttcttgagtt tgtaacagct gctgggatta cacatggcat ggatgaacta tacaaatgaa
      781 gcttggatcc ccgggaattc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
      841 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
      901 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
      961 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     1021 attgggcgct agcggagtgt atatactggc ttactatgtt ggcactgatg agggtgtcag
     1081 tgaagtgctt catgtggcag gagaaaaaag gctgcaccgg tgcgtcagca gaatatgtga
     1141 tacaggatat attccgcttc ctcgctcact gactcgctac gctcggtcgt tcgactgcgg
     1201 cgagcggaaa tggcttacga acggggcgga gatttcctgg aagatgccag gaagatactt
     1261 aacagggaag tgagagggcc gcggcaaagc cgtttttcca taggctccgc ccccctgaca
     1321 agcatcacga aatctgacgc tcaaatcagt ggtggcgaaa cccgacagga ctataaagat
     1381 accaggcgtt tccccctggc ggctccctcg tgcgctctcc tgttcctgcc tttcggttta
     1441 ccggtgtcat tccgctgtta tggccgcgtt tgtctcattc cacgcctgac actcagttcc
     1501 gggtaggcag ttcgctccaa gctggactgt atgcacgaac cccccgttca gtccgaccgc
     1561 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg aaagacatgc aaaagcacca
     1621 ctggcagcag ccactggtaa ttgatttaga ggagttagtc ttgaagtcat gcgccggtta
     1681 aggctaaact gaaaggacaa gttttggtga ctgcgctcct ccaagccagt tacctcggtt
     1741 caaagagttg gtagctcaga gaaccttcga aaaaccgccc tgcaaggcgg ttttttcgtt
     1801 ttcagagcaa gagattacgc gcagaccaaa acgatctcaa gaagatcatc ttattaatca
     1861 gataaaatat cgagccgaga acgttatcga agttggtcat gtgtaatccc ctcgtttgaa
     1921 ctttggatta agcgtagata cacccttgga caagccagtt ggattcggag acaagcaaat
     1981 tcagccttaa aaagggcgag gccctgcggt ggtggaacac cgcagggcct ctaaccgctc
     2041 gacgcgctgc accaaccagc ccgcgaacgg ctggcagcca gcgtaaggcg cggctcatcg
     2101 ggcggcgttc gccacgatgt cctgcacttc gagccaagcc tcgaacacct gctggtgtgc
     2161 acgactcacc cggttgttga caccgcgcgc ggccgtgcgg gctcggtggg gcggctctgt
     2221 cgcccttgcc agcgtgagta gcgcgtacct cacctcgccc aacaggtcgc acacagccga
     2281 ttcgtacgcc ataaagccag gtgagcccac cagctccgta agttcgggcg ctgtgtggct
     2341 cgtacccgcg cattcaggcg gcagggggtc taacgggtct aaggcggcgt gtacggccgc
     2401 cacagcggct ctcagcggcc cggaaacgtc ctcgaaacga cgcatgtgtt cctcctggtt
     2461 ggtacaggtg gttgggggtg ctcggctgtc gctggtgttc caccaccagg gctcgacggg
     2521 agagcggggg agtgtgcagt tgtggggtgg cccctcagcg aaatatctga cttggagctc
     2581 gtgtcggacc atacaccggt gattaatcgt ggtctactac caagcgtgag ccacgtcgcc
     2641 gacgaatttg agcagctctg gctgccgtac tggccgctgg caagcgacga tctgctcgag
     2701 gggatctacc gccaaagccg cgcgtcggcc ctaggccgcc ggtacatcga ggcgaaccca
     2761 acagcgctgg caaacctgct ggtcgtggac gtagaccatc cagacgcagc gctccgagcg
     2821 ctcagcgccc gggggtccca tccgctgccc aacgcgatcg tgggcaatcg cgccaacggc
     2881 cacgcacacg cagtgtgggc actcaacgcc cctgttccac gcaccgaata cgcgcggcgt
     2941 aagccgctcg catacatggc ggcgtgcgcc gaaggccttc ggcgcgccgt cgacggcgac
     3001 cgcagttact caggcctcat gaccaaaaac cccggccaca tcgcctggga aacggaatgg
     3061 ctccactcag atctctacac actcagccac atcgaggccg agctcggcgc gaacatgcca
     3121 ccgccgcgct ggcgtcagca gaccacgtac aaagcggctc cgacgccgct agggcggaat
     3181 tgcgcactgt tcgattccgt caggttgtgg gcctatcgtc ccgccctcat gcggatctac
     3241 ctgccgaccc ggaacgtgga cggactcggc cgcgcgatct atgccgagtg ccacgcgcga
     3301 aacgccgaat tcccgtgcaa cgacgtgtgt cccggaccgc taccggacag cgaggtccgc
     3361 gccatcgcca acagcatttg gcgttggatc acaaccaagt cgcgcatttg ggcggacggg
     3421 atcgtggtct acgaggccac actcagtgcg cgccagtcgg ccatctcgcg gaagggcgca
     3481 gcagcgcgca cggcggcgag cacagttgcg cggcgcgcaa agtccgcgtc agccatggag
     3541 gcattgctat gagcgacggc tacagcgacg gctacaaccg gcagccgact gtccgcaaaa
     3601 agcggcgcgt gaccgccgcc gaaggcgctc gaatcaccgg actatccgaa cgccacgtcg
     3661 tccggctcgt ggcgcaggaa cgcagcgagt ggctcgccga gcaggctgca cgccgcgaac
     3721 gcatccgcgc ctatcacgac gacgagggcc actcttggcc gcaaacggcc aaacatttcg
     3781 ggctgcatct ggacaccgtt aagcgactcg gctatcgggc gaggaaagag cgtgcggcag
     3841 aacaggaagc ggctcaaaag gcccacaacg aagccgacaa tccaccgctg ttctaacgca
     3901 attggggagc gggtgtcgcg ggggttccgt ggggggttcc gttgcaacgg gtcggacagg
     3961 taaaagtcct ggtagacgct agttttctgg tttgggccat gcctgtctcg ttgcgtgttt
     4021 cgttgcgccc gttttgaata ccagccagac gagacggggt tctacgaatc ttggtcgata
     4081 ccaagccatt tccgctgaat atcggggagc tcaccgccag aatcggtggt tgtggtgatg
     4141 tacgtggcga actccgttgt agtgcctgtg gtggcatccg tggccactct cgttgcacgg
     4201 ttcgttgtgc cgttacaggc cccgttgaca gctcaccgaa cgtagttaaa acatgctggt
     4261 caaactaggt ttaccaacga tacgagtcag ctcatctagg gccagttcta ggcgttgttc
     4321 gttgcgcggt tcgttgcgca tgtttcgtgt ggttgctaga tggctccgca accacacgct
     4381 tcgaggttga gtgcttccag cacgggcgcg atccagaaga acttcgtcgt gcgactgtcc
     4441 tcgttatttc taggcacagc ttcacgctgc cgcaagcact cagggcgcaa gggctgctaa
     4501 aggaagcgga acacgtagaa agccagtccg cagaaacggt gctgaccccg gatgaatgtc
     4561 agctactggg ctatctggac aagggaaaac gcaagcgcaa agagaaagca ggtagcttgc
     4621 agtgggctta catggcgata gctagactgg gcggttttat ggacagcaag cgaaccggaa
     4681 ttgccagctg gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa ctggatggct
     4741 ttcttgccgc caaggatctg atggcgcagg ggatcaagat ctgatcaaga gacaggatga
     4801 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     4861 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     4921 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     4981 ctgaatgaac tccaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     5041 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     5101 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     5161 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     5221 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     5281 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     5341 cggatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     5401 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     5461 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     5521 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     5581 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     5641 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     5701 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     5761 tggagttctt cgcccacccc ctgtccctcc tgttcagcta ctgacggggt ggtgcgtaac
     5821 ggcaaaagca ccgccggaca tcagcgccat tcgccattca ggctgcgcaa ctgttgggaa
     5881 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca
     5941 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc
     6001 agt
//
