LOCUS       sequence-488480-        2808 bp    DNA     circular SYN 06-OCT-2025
DEFINITION  Kanamycin/ColEI backbone with sfGFP placeholder for lvl2 assembly.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2808)
  AUTHORS   Inckemann R, Chotel T, Brinkmann CK, Burgis M, Andreas L, Baumann J,
            Sharma P, Klose M, Barrett J, Ries F, Paczia N, Glatter T, Willmund 
            F, Mackinder LCM, Erb TJ
  TITLE     Advancing chloroplast synthetic biology through high-throughput 
            plastome engineering of Chlamydomonas reinhardtii
  JOURNAL   bioRxiv 2024.05.08.593163
REFERENCE   2  (bases 1 to 2808)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 6, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2024.05.08.593163"
COMMENT     Sequence Label: pME_Cp_0_7-8_006_Kan/ColEI_sfGFP
FEATURES             Location/Qualifiers
     source          1..2808
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(19..46)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(62..133)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(145..858)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     RBS             865..876
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     rep_origin      complement(1145..1733)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1225..1244)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1947..1966)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1958..2773
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             1958..2773
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2031..2050)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 agtcttgaga cgtataaacg cagaaaggcc cacccgaagg tgagccagtg tgactctagt
       61 agagagcgtt caccgacaaa caacagataa aacgaaaggc ccagtctttc gactgagcct
      121 ttcgttttat ttgatgcctg gtcatttgta cagttcatcc ataccatgcg tgatgcccgc
      181 tgcggttacg aactccagca gaaccatatg atcgcgtttc tcgttcggat ctttagacag
      241 aacgctttgc gtgctcagat agtgattgtc tggcagcaga acaggaccat caccgattgg
      301 agtgttttgc tggtagtgat cagccagctg cacgctgcca tcctccacgt tgtggcgaat
      361 tttaaaattc gctttaatgc catttttttg tttatcggcg gtgatgtaaa cattgtggct
      421 gttaaaattg tattccagct tatggcccag gatattgccg tcctctttaa agtcaatgcc
      481 tttcagctca atgcggttta ccagggtatc gccttcaaat ttcacttccg cacgcgtttt
      541 gtacgtgccg tcatccttaa aggaaatcgt gcgttcctgc acatagcctt ccggcatggc
      601 ggacttgaag aagtcatgct gcttcatatg gtccggataa cgagcaaagc actgaacacc
      661 ataagtcagc gtcgttacca gagtcggcca aggaaccggc agtttaccag tagtacagat
      721 gaacttcagc gtcagtttac cattagttgc gtcaccttca ccctcgccac gcacggaaaa
      781 cttatgaccg ttgacatcac catccagttc caccagaata gggacgacac cagtgaacag
      841 ctcttcgcct ttacgcatct agtatttctc ctctttctct agtagctaag atccgaacct
      901 gccgtttctt gagttgccgc gatgttaaga aaacattcat aaattaaatg tgaattgccg
      961 cacacattat taaataagat ttacaaaatg ttcaaaatga cgcatgaaat cacgtttcac
     1021 tttcctcgtc tcatgttgcg gccgcacggt tatccacaga atcaggggat aacgcaggaa
     1081 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     1141 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     1201 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     1261 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     1321 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     1381 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     1441 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     1501 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     1561 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     1621 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     1681 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     1741 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     1801 tggtcatgac tgatccttca actcagcaaa agttcgattt attcaacaaa gccacgttgt
     1861 gtctcaaaat ctctgatgtt acattgcaca agataaaaat atatcatcat gaacaataaa
     1921 actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac
     1981 gtcttgctcc aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg
     2041 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga
     2101 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga
     2161 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat
     2221 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca
     2281 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct
     2341 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg
     2401 cctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga
     2461 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataagc ttttgccatt
     2521 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga
     2581 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga
     2641 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt
     2701 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga
     2761 tgagtttttc taatcagaat tggttaattg gttgtaacac gcggccgc
//
