LOCUS       sequence-413934-        3556 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Creation of Level 0 entry clones.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3556)
  AUTHORS   Piepers M, Erbstein K, Reyes-Hernandez J, Song C, Tessi T, 
            Petrasiunaite V, Faerber N, Distel K, Maizel A
  TITLE     GreenGate 2.0: Backwards compatible addons for assembly of complex 
            transcriptional units and their stacking with GreenGate.
  JOURNAL   PLoS One. 2023 Sep 8;18(9):e0290097. doi: 
            10.1371/journal.pone.0290097. eCollection 2023.
  PUBMED    37682951
REFERENCE   2  (bases 1 to 3556)
  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: "PLoS One.";
            date: "2023-09-8"; volume: "18(9):e0290097. doi"; pages: " 
            10.1371/journal.pone.0290097. eCollection 2023"
COMMENT     Sequence Label: pR562
FEATURES             Location/Qualifiers
     source          1..3556
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     379..401
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     393..410
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     394..410
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     421..440
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        421..439
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    606..627
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(642..647,648..665,666..672)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    680..696
                     /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             714..725
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      1442..1513
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1529..1556
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(1713..1730)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      1767..1810
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(1867..2455)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1947..1966)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2626..3417,3418..3486))
                     /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     3249..3268
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3487..3550)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gctaataaca ggcctgctgg taatcgcagg cctttttatt tggtgtattt agaaaaataa
       61 acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat
      121 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
      181 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtggccgc
      241 tacagggcgc tcccattcgc cattcaggct gcgcaactgt tgggaagggc gtttcggtgc
      301 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt
      361 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt gagcgcgacg
      421 taatacgact cactataggg cgaattgtag gcggaaggcc gtcaaggcct aggcgcgcca
      481 tgagctcggt ctcattgtgc aggtgcaata cgcaaaccgc ctctccccgc gcgttggccg
      541 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
      601 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
      661 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacatacta gagaaagagg
      721 agaaatacta gatgagtgtg atcgctaaac aaatgaccta caaggtttat atgtcaggca
      781 cggtcaatgg acactacttt gaggtcgaag gcgatggaaa aggtaagccc tacgaggggg
      841 agcagacggt aaagctcact gtcaccaagg gcggacctct gccatttgct tgggatattt
      901 tatcaccaca gtgtcagtac ggaagcatac cattcaccaa gtaccctgaa gacatccctg
      961 actatgtaaa gcagtcattc ccggagggct atacatggga gaggatcatg aactttgaag
     1021 atggtgcagt gtgtactgtc agcaatgatt ccagcatcca aggcaactgt ttcatctacc
     1081 atgtcaagtt ctctggtttg aactttcctc ccaatggacc tgtcatgcag aagaagacac
     1141 agggctggga acccaacact gagcgtctct ttgcacgaga tggaatgctg ctaggaaaca
     1201 actttatggc tctgaagtta gaaggaggcg gtcactattt gtgtgaattt aaaactactt
     1261 acaaggcaaa gaagcctgtg aagatgccag ggtatcacta tgttgaccgc aaactggatg
     1321 taaccaatca caacaaggat tacacttcgg ttgagcagtg tgaaatttcc attgcacgca
     1381 aacctgtggt cgcctaataa cgctgatagt gctagtgtag atcgctacta gagccaggca
     1441 tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc
     1501 ggtgaacgct ctctactaga gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta
     1561 tacacctgca caacgagacc ggtacctctt aattaactgg cctcatgggc cttccgccta
     1621 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaaca tggtcatagc
     1681 tgtttccttg cgtattgggc gctctccgct tcctcgctca ctgactcgct gcgctcggtc
     1741 gttcgggtaa agcctggggt gcctaaataa aacgaaaggc ccagtcttcc gactgagcct
     1801 ttcgttttat tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     1861 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     1921 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     1981 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     2041 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     2101 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     2161 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     2221 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     2281 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     2341 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     2401 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     2461 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     2521 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     2581 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     2641 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     2701 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     2761 accgcgagaa ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     2821 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     2881 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     2941 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     3001 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     3061 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     3121 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     3181 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     3241 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     3301 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     3361 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     3421 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     3481 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     3541 cggatacata tttgaa
//
