LOCUS       sequence-149663-        6674 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GentR version of pRIM2 carrying rfp.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6674)
  AUTHORS   Cleto S, Jensen JV, Wendisch VF, Lu TK
  TITLE     Corynebacterium glutamicum Metabolic Engineering with CRISPR 
            Interference (CRISPRi).
  JOURNAL   ACS Synth Biol. 2016 Feb 16.
  PUBMED    26829286
REFERENCE   2  (bases 1 to 6674)
  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. 2016 Feb 16."
COMMENT     Sequence Label: pRIM3-rfp
FEATURES             Location/Qualifiers
     source          1..6674
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             108..767
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(174..193)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1203..1289
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1381..1408
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     oriT            1751..1860
                     /note="incP origin of transfer"
     primer_bind     complement(2723..2740)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2723..2739)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2732..2754)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(3289..3294,3295..3311,3312..3317)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             3506..4039
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     rep_origin      4354..4942
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4843..4862
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5096..5113
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(5227..5232,5233..5248,5249..5255)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    5263..5279
                     /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)."
     CDS             5315..5992
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      6218..6304
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6396..6423
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     protein_bind    6548..6569
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6584..6589,6590..6607,6608..6614)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6622..6638
                     /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     6646..6662
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6646..6662
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 aagcttgcat gcctgcaggt cgactctaga ggatccccgg gtacccagct tgagtaggac
       61 aaatccgccg agcttcgacg agattttcag gagctaagga agctaaaatg gagaaaaaaa
      121 tcactggata taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat
      181 ttcagtcagt tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt
      241 taaagaccgt aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc
      301 gcctgatgaa tgctcatccg gaattccgta tggcaatgaa agacggtgag ctggtgatat
      361 gggatagtgt tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc
      421 tctggagtga ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg
      481 cgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg
      541 tctcagccaa tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca
      601 acttcttcgc ccccgttttc accatgggca aatattatac gcaaggcgac aaggtgctga
      661 tgccgctggc gattcaggtt catcatgccg tttgtgatgg cttccatgtc ggcagaatgc
      721 ttaatgaatt acaacagtac tgcgatgagt ggcagggcgg ggcgtaattt ttttaaggca
      781 gttattggtg cccttaaacg cctggttgct acgcctgaat aagtgataat aagcggatga
      841 atggcagaaa ttcgtcgagg cggcacctcg ctaacggatt caccactcca agaattggag
      901 ccaatcaatt cttgcggaga actgtgaatg cgcaaaccaa cccttggcag aacatatcca
      961 tcgcgtccgc catctccagc agccgcacgc ggcgcatctc ggctgttttg gcggatgaga
     1021 gaagattttc agcctgatac agattaaatc agaacgcaga agcggtctga taaaacagaa
     1081 tttgcctggc ggcagtagcg cggtggtccc acctgacccc atgccgaact cagaagtgaa
     1141 acgccgtagc gccgatggta gtgtggggtc tccccatgcg agagtaggga actgccaggc
     1201 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt
     1261 cggtgaacgc tctcctgagt aggacaaatc cgccgggagc ggatttgaac gttgcgaagc
     1321 aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggcat caaattaagc
     1381 agaaggccat cctgacggat ggcctttttg cgtttctaca aactcttcct gtcgtcatat
     1441 ctacaagcca tccccccaca gatacggtaa actagcctcg tttttgcatc aggaaagcag
     1501 ggggatcgat ccccctcttt ggcatcgtct ctcgcctgtc ccctcagttc agtaatttcc
     1561 tgcatttgcc tgtttccagt cggtagatat tccacaaaac agcagggaag cagcgctttt
     1621 ccgctgcata accctgcttc ggggtcatta tagcgatttt ttcggtatat ccatcctttt
     1681 tcgcacgata tacaggattt tgccaaaggg ttcgtgtaga ctttccttgg tgtatccaac
     1741 ggcgtcagcc gggcaggata ggtgaagtag gcccacccgc gagcgggtgt tccttcttca
     1801 ctgtccctta ttcgcacctg gcggtgctca acgggaatcc tgctctgcga ggctggccgg
     1861 ctaccgccgg cgtaacagat gagggcaagc ggatggctga tgaaaccaag ccaaccagga
     1921 agggcagccc acctatcaag gtgtactgcc ttccagacga acgaagagcg attgaggaaa
     1981 aggcggcggc ggccggcatg agcctgtcgg cctacctgct ggccgtcggc cagggctaca
     2041 aaatcacggg cgtcgtggac tatgagcacg tccgcgaggg ctggctgggt agtactcgtg
     2101 tatactgtct aaagttattc gaaatcaggt gggcataagg ttcacctggg ttctcaaacg
     2161 gcaaaggaac attttccaca tggcattgac gcttcaaatc atcctcgtcg tcgccagcct
     2221 gctcatgacg gttttcgtct tgctgcacaa gggcaaaggc ggcggactct ccagcctctt
     2281 cggtggcggt gtgcagtcca atctttcggg ctccactgtt gttgaaaaga acctggatcg
     2341 cgtcaccatt ttggttgccg ttatctggat tgtgtgcatt gtcgcactca acctcatcca
     2401 gacttattca taagacacga gcttaaaaag agcggttccc ttttcatagg ggagccgctt
     2461 ttttgggttt tgtcgacctg ttgtctcccc actgttcctc ggtgtgcact ttcgacacca
     2521 agatttcggc aaagtggtgg tcaaaattgg aaaatcttgg tgcctaattc acatacattc
     2581 caattttccc taaggacatc tttaaagggg aactgtttcc cgacggaacg tggagtctat
     2641 aaaaccgcag gttaaaacgc tgccataggg gatttttcgg ctggggagac gtggtgtaag
     2701 tgcgggaatt cgagctcgat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     2761 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     2821 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc
     2881 gataagctag cttcacgctg ccgcaagcac tcagggcgca agggctgcta aaggaagcgg
     2941 aacacgtaga aagccagtcc gcagaaacgg tgctgacccc ggatgaatgt cagctactgg
     3001 gctatctgga caagggaaaa cgcaagcgca aagagaaagc aggtagcttg cagtgggctt
     3061 acatggcgat agctagactg ggcggtttta tggacagcaa gcgaaccgga attgccagct
     3121 ggggcgccct ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttcttgccg
     3181 ccaaggatct gatggcgcag gggatcaaga tctgatcaag agacaggatg aggatcgttt
     3241 cgcatgattg aacaagatgg attgcacgca ggttctccaa ttctcgaatt gacataagcc
     3301 tgttcggttc gtaaactgta atgcaagtag cgtatgcgct cacgcaactg gtccagaacc
     3361 ttgaccgaac gcagcggtgg taacggcgca gtggcggttt tcatggcttg ttatgactgt
     3421 ttttttgtac agtctatgcc tcgggcatcc aagcagcaag cgcgttacgc cgtgggtcga
     3481 tgtttgatgt tatggagcag caacgatgtt acgcagcagc aacgatgtta cgcagcaggg
     3541 cagtcgccct aaaacaaagt taggtggctc aagtatgggc atcattcgca catgtaggct
     3601 cggccctgac caagtcaaat ccatgcgggc tgctcttgat cttttcggtc gtgagttcgg
     3661 agacgtagcc acctactccc aacatcagcc ggactccgat tacctcggga acttgctccg
     3721 tagtaagaca ttcatcgcgc ttgctgcctt cgaccaagaa gcggttgttg gcgctctcgc
     3781 ggcttacgtt ctgcccaagt ttgagcagcc gcgtagtgag atctatatct atgatctcgc
     3841 agtctccggc gagcaccgga ggcagggcat tgccaccgcg ctcatcaatc tcctcaagca
     3901 tgaggccaac gcgcttggtg cttatgtgat ctacgtgcaa gcagattacg gtgacgatcc
     3961 cgcagtggct ctctatacaa agttgggcat acgggaagaa gtgatgcact ttgatatcga
     4021 cccaagtacc gccacctaac aattcgttca agccgagatc ggcttcccgg gcgggactct
     4081 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
     4141 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     4201 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaaaagga tctaggtgaa
     4261 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     4321 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     4381 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     4441 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     4501 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     4561 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     4621 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     4681 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     4741 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     4801 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     4861 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     4921 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     4981 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     5041 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     5101 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     5161 gccgattcat taatgcagct tgcgccgaca tcataacggt tctggcaaat attctgaaat
     5221 gagctgttga caattaatca tcggctcgta taatgtgtgg aattgtgagc ggataacaat
     5281 ttcacacagg aaacagaatt gaaaggagga caccatggcg agtagcgaag acgttatcaa
     5341 agagttcatg cgtttcaaag ttcgtatgga aggttccgtt aacggtcacg agttcgaaat
     5401 cgaaggtgaa ggtgaaggtc gtccgtacga aggtacccag accgctaaac tgaaagttac
     5461 caaaggtggt ccgctgccgt tcgcttggga catcctgtcc ccgcagttcc agtacggttc
     5521 caaagcttac gttaaacacc cggctgacat cccggactac ctgaaactgt ccttcccgga
     5581 aggtttcaaa tgggaacgtg ttatgaactt cgaagacggt ggtgttgtta ccgttaccca
     5641 ggactcctcc ctgcaagacg gtgagttcat ctacaaagtt aaactgcgtg gtaccaactt
     5701 cccgtccgac ggtccggtta tgcagaaaaa aaccatgggt tgggaagctt ccaccgaacg
     5761 tatgtacccg gaagacggtg ctctgaaagg tgaaatcaaa atgcgtctga aactgaaaga
     5821 cggtggtcac tacgacgctg aagttaaaac cacctacatg gctaaaaaac cggttcagct
     5881 gccgggtgct tacaaaaccg acatcaaact ggacatcacc tcccacaacg aagactacac
     5941 catcgttgaa cagtacgaac gtgctgaagg tcgtcactcc accggtgctt aagatccgtc
     6001 gacctgcagc caagcttctg ttttggcgga tgagagaaga ttttcagcct gatacagatt
     6061 aaatcagaac gcagaagcgg tctgataaaa cagaatttgc ctggcggcag tagcgcggtg
     6121 gtcccacctg accccatgcc gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg
     6181 gggtctcccc atgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc
     6241 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac
     6301 aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg
     6361 acgcccgcca taaactgcca ggcatcaaat taagcagaag gccatcctga cggatggcct
     6421 ttttgcgttt ctacaaactc ttttgtttat ttttctaaat acattcaaat atgtatccgc
     6481 tcatgagaca ataaccctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     6541 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     6601 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     6661 accatgatta cgcc
//
