LOCUS       sequence-496059-        5464 bp    DNA     circular SYN 02-JAN-2026
DEFINITION  Level 1 Position 2 FORWARD acceptor with Amil RFP negative selection
            cassette GGAG - GCTT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5464)
  TITLE     Plant Golden-gate Promoters, Terminators and Level0/Level1 cloning 
            acceptors.
REFERENCE   2  (bases 1 to 5464)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 2, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pICSL47742_RFP
FEATURES             Location/Qualifiers
     source          1..5464
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    146..167
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(182..187,188..205,206..212)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    220..236
                     /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             254..265
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             272..949
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      994..1065
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1081..1108
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    1167..1191
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(1347..2495)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     rep_origin      complement(2831..3419)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2911..2930)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3590..4381,4382..4450))
                     /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     4213..4232
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4451..4555)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4642..5273
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    5365..5389
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 tctgtgaaga caagcaagaa ttcaagcttg gagtgagacc gatatcaata cgcaaaccgc
       61 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
      121 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
      181 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
      241 acacatacta gagaaagagg agaaatacta gatggcttcc tccgaggacg ttatcaaaga
      301 gttcatgcgt ttcaaagttc gtatggaagg ttccgttaac ggtcacgagt tcgaaatcga
      361 aggtgaaggt gaaggtcgtc cgtacgaagg tacccagacc gctaaactga aagttaccaa
      421 aggtggtccg ctgccgttcg cttgggacat cctgtccccg cagttccagt acggttccaa
      481 agcttacgtt aaacacccgg ctgacatccc ggactacctg aaactgtcct tcccggaagg
      541 tttcaaatgg gaacgtgtta tgaacttcga ggacggtggt gttgttaccg ttacccagga
      601 ctcctccctg caagacggtg agttcatcta caaagttaaa ctgcgtggta ccaacttccc
      661 gtccgacggt ccggttatgc agaaaaaaac catgggttgg gaagcttcca ccgaacgtat
      721 gtacccggag gacggtgctc tgaaaggtga aatcaaaatg cgtctgaaac tgaaagacgg
      781 tggtcactac gacgctgaag ttaaaaccac ctacatggct aaaaaaccgg ttcagctgcc
      841 gggtgcttac aaaaccgaca tcaaactgga catcacctcc cacaacgagg actacaccat
      901 cgttgaacag tacgaacgtg ctgaaggtcg tcactccacc ggtgcttaat aacgctgata
      961 gtgctagtgt agatcgctac tagagccagg catcaaataa aacgaaaggc tcagtcgaaa
     1021 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctctacta gagtcacact
     1081 ggctcacctt cgggtgggcc tttctgcgtt tataggtctc acgctactat tgtcttctgc
     1141 acgaagtggt ttaaactatc agtgtttgac aggatatatt ggcgggtaaa cctaagagaa
     1201 aagagcgttt attagaataa tcggatattt aaaagggcgt gaaaaggttt atccgttcgt
     1261 ccatttgtat gtgcatgcca accacagggt tccccagatc aggcgctggc tgctgaaccc
     1321 ccagccggaa ctgaccccac aaggccctag cgtttgcaat gcaccaggtc atcattgacc
     1381 caggcgtgtt ccaccaggcc gctgcctcgc aactcttcgc aggcttcgcc gacctgctcg
     1441 cgccacttct tcacgcgggt ggaatccgat ccgcacatga ggcggaaggt ttccagcttg
     1501 agcgggtacg gctcccggtg cgagctgaaa tagtcgaaca tccgtcgggc cgtcggcgac
     1561 agcttgcggt acttctccca tatgaatttc gtgtagtggt cgccagcaaa cagcacgacg
     1621 atttcctcgt cgatcaggac ctggcaacgg gacgttttct tgccacggtc caggacgcgg
     1681 aagcggtgca gcagcgacac cgattccagg tgcccaacgc ggtcggacgt gaagcccatc
     1741 gccgtcgcct gtaggcgcga caggcattcc tcggccttcg tgtaataccg gccattgatc
     1801 gaccagccca ggtcctggca aagctcgtag aacgtgaagg tgatcggctc gccgataggg
     1861 gtgcgcttcg cgtactccaa cacctgctgc cacaccagtt cgtcatcgtc ggcccgcagc
     1921 tcgacgccgg tgtaggtgat cttcacgtcc ttgttgacgt ggaaaatgac cttgttttgc
     1981 agcgcctcgc gcgggatttt cttgttgcgc gtggtgaaca gggcagagcg ggccgtgtcg
     2041 tttggcatcg ctcgcatcgt gtccggccac ggcgcaatat cgaacaagga aagctgcatt
     2101 tccttgatct gctgcttcgt gtgtttcagc aacgcggcct gcttggcctc gctgacctgt
     2161 tttgccaggt cctcgccggc ggtttttcgc ttcttggtcg tcatagttcc tcgcgtgtcg
     2221 atggtcatcg acttcgccaa acctgccgcc tcctgttcaa gacgacgcga acgctccacg
     2281 gcggccgatg gcgcgggcag ggcaggggga gccagttgca cgctgtcgcg ctcgatcttg
     2341 gccgtagctt gctggaccat cgagccgacg gactggaagg tttcgcgggg cgcacgcatg
     2401 acggtgcggc ttgcgatggt ttcggcatcc tcggcggaaa accccgcgtc gatcagttct
     2461 tgcctgtatg ccttccggtc aaacgtccga ttcattcacc ctccttgcgg gattgccccg
     2521 actcacgccg gggcaatgtg cccttattcc tgatttgacc cgcctggtgc cttggtgtcc
     2581 agataatcca ccttatcggc aatgaagtcg gtcccgtaga ccgtctggcc gtccttctcg
     2641 tacttggtat tccgaatctt gccctgcacg aataccagcg accccttgcc caaatacttg
     2701 ccgtgggcct cggcctgaga gccaaaacac ttgatgcgga agaagtcggt gcgctcctgc
     2761 ttgtcgccgg catcgttgcg ccacatctag gatctgccag gaaccgtaaa aaggccgcgt
     2821 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     2881 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     2941 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     3001 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     3061 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     3121 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     3181 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     3241 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     3301 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     3361 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     3421 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     3481 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     3541 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     3601 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     3661 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     3721 tgataccgcg agaaccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     3781 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     3841 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     3901 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     3961 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     4021 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     4081 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     4141 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     4201 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     4261 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     4321 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     4381 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     4441 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcaaggt tattgtctca
     4501 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacga
     4561 attggccagc gctgccattt ttggggtgag gccgttcgcg gccgaggggc gcagcccctg
     4621 gggggatggg aggcccgcgt tagcgggccg ggagggttcg agaagggggg gcacccccct
     4681 tcggcgtgcg cggtcacgcg cacagggcgc agccctggtt aaaaacaagg tttataaata
     4741 ttggtttaaa agcaggttaa aagacaggtt agcggtggcc gaaaaacggg cggaaaccct
     4801 tgcaaatgct ggattttctg cctgtggaca gcccctcaaa tgtcaatagg tgcgcccctc
     4861 atctgtcagc actctgcccc tcaagtgtca aggatcgcgc ccctcatctg tcagtagtcg
     4921 cgcccctcaa gtgtcaatac cgcagggcac ttatccccag gcttgtccac atcatctgtg
     4981 ggaaactcgc gtaaaatcag gcgttttcgc cgatttgcga ggctggccag ctccacgtcg
     5041 ccggccgaaa tcgagcctgc ccctcatctg tcaacgccgc gccgggtgag tcggcccctc
     5101 aagtgtcaac gtccgcccct catctgtcag tgagggccaa gttttccgcg aggtatccac
     5161 aacgccggcg gccgcggtgt ctcgcacacg gcttcgacgg cgtttctggc gcgtttgcag
     5221 ggccatagac ggccgccagc ccagcggcga gggcaaccag cccggtgagc gtcgcaaagg
     5281 agatcctgat ctgactgatg ggctgcctgt atcgagtggt gattttgtgc cgagctgccg
     5341 gtcggggagc tgttggctgg ctggtggcag gatatattgt ggtgtaaaca aattgacgct
     5401 tagacaactt aataacacat tgcggacgtt tttaatgtac tggggtggat gcagtgggcc
     5461 ccac
//
