LOCUS       sequence-463319-        5319 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains GFPmut3b under the Ptrc promoter. Suicide vector inserts 
            into slr0168 site of Synechocystis. Carries chloramphenicol 
            resistance. Propogates in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5319)
  AUTHORS   Yao L, Cengic I, Anfelt J, Hudson EP
  TITLE     Multiple Gene Repression in Cyanobacteria Using CRISPRi.
  JOURNAL   ACS Synth Biol. 2015 Dec 28.
  PUBMED    26689101
REFERENCE   2  (bases 1 to 5319)
  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: "ACS Synth 
            Biol. 2015 Dec 28."
COMMENT     Sequence Label: pMD19T-slr0168-Ptrc-GFP3mut-CmR
FEATURES             Location/Qualifiers
     source          1..5319
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(504..509,510..526,527..533)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    541..557
                     /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             567..578
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             584..1297
                     /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="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(614..642)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1237..1257
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      1320..1391
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1407..1434
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             1542..2201
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1608..1627)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     misc_feature    2202..2222
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     promoter        complement(join(3200..3206,3207..3224,3225..3230))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3245..3266
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3383..3400)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3554..4142)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3634..3653)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4313..5104,5105..5173))
                     /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     4936..4955
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5174..5278)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggcgttaatc tggaaaaaaa cgataacatt cccattgttt ttgcgtggcc catttttccc
       61 accaccctta atcccacaga ttttcaggta atgcttaaca cgggggaaat tgtcaccccg
      121 gtgatcgcct ctttgattcc caacagtgaa tacaacgaac ggcaaacggt agtaattacg
      181 ggcaattttg gtaatcgttt aaccccaggc acggagggag cgatttatcc cgtttccgta
      241 ggcacagtgt tggacagtac tcctttggaa atggtgggac ccaacggccc ggtcagtgcg
      301 gtgggtatta ccattgatag tctcaacccc tacgtggccg gcaatggtcc caaaattgtc
      361 gccgctaagt tagaccgctt cagtgacctg ggggaagggg ctcccctctg gttagccacc
      421 aatcaaaata acagtggcgg ggatttatat ggagaccaag cccaatttcg tttgcgaatt
      481 cgcggccgct ctagaatgag ctgttgacaa ttaatcatcc ggctcgtata atgtgtggaa
      541 ttgtgagcgg ataacaattt cacacaaaag aggagaaaac tagatgcgta aaggagaaga
      601 acttttcact ggagttgtcc caattcttgt tgaattagat ggtgatgtta atgggcacaa
      661 attttctgtc agtggagagg gtgaaggtga tgcaacatac ggaaaactta cccttaaatt
      721 tatttgcact actggaaaac tacctgttcc atggccaaca cttgtcacta ctttcggtta
      781 tggtgttcaa tgctttgcga gatacccaga tcatatgaaa cagcatgact ttttcaagag
      841 tgccatgccc gaaggttatg tacaggaaag aactatattt ttcaaagatg acgggaacta
      901 caagacacgt gctgaagtca agtttgaagg tgataccctt gttaatagaa tcgagttaaa
      961 aggtattgat tttaaagaag atggaaacat tcttggacac aaattggaat acaactataa
     1021 ctcacacaat gtatacatca tggcagacaa acaaaagaat ggaatcaaag ttaacttcaa
     1081 aattagacac aacattgaag atggaagcgt tcaactagca gaccattatc aacaaaatac
     1141 tccaattggc gatggccctg tccttttacc agacaaccat tacctgtcca cacaatctgc
     1201 cctttcgaaa gatcccaacg aaaagagaga ccacatggtc cttcttgagt ttgtaacagc
     1261 tgctgggatt acacatggca tggatgaact atacaaataa taatactaga gccaggcatc
     1321 aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg
     1381 tgaacgctct ctactagagt cacactggct caccttcggg tgggcctttc tgcgtttata
     1441 tactagaggt aagaggttcc aactttcacc ataatgaaat aagatcacta ccgggcgtat
     1501 tttttgagtt atcgagattt tcaggagcta aggaagctaa aatggagaaa aaaatcactg
     1561 gatataccac cgttgatata tcccaatggc atcgtaaaga acattttgag gcatttcagt
     1621 cagttgctca atgtacctat aaccagaccg ttcagctgga tattacggcc tttttaaaga
     1681 ccgtaaagaa aaataagcac aagttttatc cggcctttat tcacattctt gcccgcctga
     1741 tgaatgctca tccggaattt cgtatggcaa tgaaagacgg tgagctggtg atatgggata
     1801 gtgttcaccc ttgttacacc gttttccatg agcaaactga aacgttttca tcgctctgga
     1861 gtgaatacca cgacgatttc cggcagtttc tacacatata ttcgcaagat gtggcgtgtt
     1921 acggtgaaaa cctggcctat ttccctaaag ggtttattga gaatatgttt ttcgtctcag
     1981 ccaatccctg ggtgagtttc accagttttg atttaaacgt ggccaatatg gacaacttct
     2041 tcgcccccgt tttcaccatg ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc
     2101 tggcgattca ggttcatcat gccgtttgtg atggcttcca tgtcggcaga atgcttaatg
     2161 aattacaaca gtactgcgat gagtggcagg gcggggcgta atactagtag cggccgctgc
     2221 aggattattc ggcggtttat aatcccggtg gccccggcaa tgatccagag aatggtcccc
     2281 cagggccctt tactgtgtcc agtagtcccc aggtaattga ggtaacggat accatcggcc
     2341 agcccaccaa agtctcctat gtggaagtgg atggccccgt attgcgtaat cccttcagtg
     2401 gtactcccat tgggcaagag gtgggtttag cggttaaaga tctggccaca ggtcatgaaa
     2461 tttatcagta cactgaccca gatgggaagg tattttatgc ttcctttgct gccgctgatg
     2521 accaagccac ggatttaacc acggcgatcg ccaatcccac ggccatcgat ttaattaacg
     2581 ccaggggatt tacggcgggt agttccgtca ccgtatcggg ttcctacagt cgggaagcct
     2641 tttttgatgg atccatgggt ttttatcgac ttctggacga taacggtgca gtgctagatc
     2701 ccttaacagg tggtgtaatc aacccaggac aggtaggtta tcaagaagca gctttggcag
     2761 atagcaatcg tttgcaagcc actggctcca ccctaacggc agaagaccta gaaaccagag
     2821 cattttcctt caatattttg ggtggcgagt tgtatgcgcc atttttaacg gttaatgaca
     2881 gtctttccgg tattaatcag acttattttg cctttgggtc ggccaaccca gatggcatca
     2941 gccacagcac aaacttggga cccaacgtga ttggttttga agattttctc ggcggaggag
     3001 acctagattt cgacgacatc attgtcagat ttacgctgac ttagctcagg ggcattatcg
     3061 gagcaagccg atattcaggt aagatggggc ggtgcttttg tgggttgatc cattagcttt
     3121 aaccccattg tctccactga cggtaatagt tttccccact gcgattaatt gttttgtttg
     3181 gaggagggac tgctccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     3241 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     3301 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     3361 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     3421 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     3481 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     3541 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     3601 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     3661 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     3721 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     3781 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     3841 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     3901 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     3961 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     4021 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4081 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4141 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4201 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     4261 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     4321 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     4381 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     4441 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     4501 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     4561 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     4621 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     4681 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     4741 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     4801 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     4861 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     4921 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     4981 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     5041 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     5101 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     5161 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     5221 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaatgggg gttccgcgca
     5281 catttccccg aaaagtgcca cctgacgtct aagaaacca
//
