LOCUS       sequence-135056-        5628 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 5628)
  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 5628)
  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..5628
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(813..818,819..835,836..842)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    850..866
                     /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             876..887
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             893..1606
                     /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(923..951)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1546..1566
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      1629..1700
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1716..1743
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             1851..2510
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1917..1936)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     misc_feature    2511..2531
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     promoter        complement(join(3509..3515,3516..3533,3534..3539))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3554..3575
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3692..3709)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3863..4451)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3943..3962)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4622..5413,5414..5482))
                     /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     5245..5264
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5483..5587)
                     /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 tacaccagcg ccggtttttc ccccgatggc attgccagtt tactacccac agaatttgaa
      541 cggtattttc aactccaagc ggaagatatt acgggacgga cagttatcct aacccaaact
      601 ggtgttgatt atgaaattcc cggctttggt ctggtgcagg tgttggggct ggcggatttg
      661 gccggggttc aggacagcta tgacctgact tacatcgaag atcatgacaa ctattacgac
      721 attatcctca aaggggacga agccgcagtt cgccaaatta agagggttgc tttgccctcc
      781 gaaggaattc gcggccgctc tagaatgagc tgttgacaat taatcatccg gctcgtataa
      841 tgtgtggaat tgtgagcgga taacaatttc acacaaaaga ggagaaaact agatgcgtaa
      901 aggagaagaa cttttcactg gagttgtccc aattcttgtt gaattagatg gtgatgttaa
      961 tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat gcaacatacg gaaaacttac
     1021 ccttaaattt atttgcacta ctggaaaact acctgttcca tggccaacac ttgtcactac
     1081 tttcggttat ggtgttcaat gctttgcgag atacccagat catatgaaac agcatgactt
     1141 tttcaagagt gccatgcccg aaggttatgt acaggaaaga actatatttt tcaaagatga
     1201 cgggaactac aagacacgtg ctgaagtcaa gtttgaaggt gatacccttg ttaatagaat
     1261 cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt cttggacaca aattggaata
     1321 caactataac tcacacaatg tatacatcat ggcagacaaa caaaagaatg gaatcaaagt
     1381 taacttcaaa attagacaca acattgaaga tggaagcgtt caactagcag accattatca
     1441 acaaaatact ccaattggcg atggccctgt ccttttacca gacaaccatt acctgtccac
     1501 acaatctgcc ctttcgaaag atcccaacga aaagagagac cacatggtcc ttcttgagtt
     1561 tgtaacagct gctgggatta cacatggcat ggatgaacta tacaaataat aatactagag
     1621 ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     1681 gtttgtcggt gaacgctctc tactagagtc acactggctc accttcgggt gggcctttct
     1741 gcgtttatat actagaggta agaggttcca actttcacca taatgaaata agatcactac
     1801 cgggcgtatt ttttgagtta tcgagatttt caggagctaa ggaagctaaa atggagaaaa
     1861 aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa cattttgagg
     1921 catttcagtc agttgctcaa tgtacctata accagaccgt tcagctggat attacggcct
     1981 ttttaaagac cgtaaagaaa aataagcaca agttttatcc ggcctttatt cacattcttg
     2041 cccgcctgat gaatgctcat ccggaatttc gtatggcaat gaaagacggt gagctggtga
     2101 tatgggatag tgttcaccct tgttacaccg ttttccatga gcaaactgaa acgttttcat
     2161 cgctctggag tgaataccac gacgatttcc ggcagtttct acacatatat tcgcaagatg
     2221 tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg gtttattgag aatatgtttt
     2281 tcgtctcagc caatccctgg gtgagtttca ccagttttga tttaaacgtg gccaatatgg
     2341 acaacttctt cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc
     2401 tgatgccgct ggcgattcag gttcatcatg ccgtttgtga tggcttccat gtcggcagaa
     2461 tgcttaatga attacaacag tactgcgatg agtggcaggg cggggcgtaa tactagtagc
     2521 ggccgctgca ggattattcg gcggtttata atcccggtgg ccccggcaat gatccagaga
     2581 atggtccccc agggcccttt actgtgtcca gtagtcccca ggtaattaag gtaacggata
     2641 ccatcggcca gcccaccaaa gtctcctatg tggaagtgga tggccccgta ttgcgtaatc
     2701 ccttcagtgg tactcccatt gggcaagagg tgggtttagc ggttaaagat ctggccacag
     2761 gtcatgaaat ttatcagtac actgacccag atgggaaggt attttatgct tcctttgctg
     2821 ccgctgatga ccaagccacg gatttaacca cggcgatcgc caatcccacg gccatcgatt
     2881 taattaacgc caggggattt acggcgggta gttccgtcac cgtatcgggt tcctacagtc
     2941 gggaagcctt ttttgatgga tccatgggtt tttatcgact tctggacgat aacggtgcag
     3001 tgctagatcc cttaacaggt ggtgtaatca acccaggaca ggtaggttat caagaagcag
     3061 ctttggcaga tagcaatcgt ttgcaagcca ctggctccac cctaacggca gaagacctag
     3121 aaaccagagc attttccttc aatattttgg gtggcgagtt gtatgcgcca tttttaacgg
     3181 ttaatgacag tctttccggt attaatcaga cttattttgc ctttgggtcg gccaacccag
     3241 atggcatcag ccacagcaca aacttgggac ccaacgtgat tggttttgaa gattttctcg
     3301 gcggaggaga cctagatttc gacgacatca ttgtcagatt tacgctgact tagctcaggg
     3361 gcattatcgg agcaagccga tattcaggta agatggggcg gtgcttttgt gggttgatcc
     3421 attagcttta accccattgt ctccactgac ggtaatagtt ttccccactg cgattaattg
     3481 ttttgtttgg aggagggact gctccacaca acatacgagc cggaagcata aagtgtaaag
     3541 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     3601 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     3661 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     3721 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     3781 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     3841 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     3901 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     3961 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4021 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4081 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4141 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4201 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     4261 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     4321 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4381 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4441 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4501 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     4561 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     4621 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     4681 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     4741 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     4801 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     4861 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     4921 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     4981 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     5041 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     5101 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     5161 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     5221 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     5281 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     5341 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     5401 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     5461 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     5521 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatggggg
     5581 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaacca
//
