LOCUS       sequence-152045-        4582 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  DEST vector for cell-free in vitro expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4582)
  TITLE     pAM-PAT-ProUBQ10 GW
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4582)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pOLENTE
FEATURES             Location/Qualifiers
     source          1..4582
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        16..34
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        40..58
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     40..57
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    124..248
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(273..278,279..296,297..303)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             357..1016
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(423..442)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1358..1663
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1542..1561
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1704..1828)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      1872..1919
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1998..2453
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2085..2104)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2295..2316
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2520..2539)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2639..2661
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2699..2717)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2785..2889
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2890..2958,2959..3750)
                     /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     complement(3108..3127)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3921..4509
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4410..4429
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 taaggctaga gtacttaata cgactcacta taggctagca tttaggtgac actatagaat
       61 acaagctact tgttcttttt gcactcgaga attcacgcgt ggtacctcta gagtcgaccc
      121 atcacaagtt tgtacaaaaa agctgaacga gaaacgtaaa atgatataaa tatcaatata
      181 ttaaattaga ttttgcataa aaaacagact acataatact gtaaaacaca acatatccag
      241 tcactatggc ggccgcatta ggcaccccag gctttacact ttatgcttcc ggctcgtata
      301 atgtgtggat tttgagttag gatccgtcga gattttcagg agctaaggaa gctaaaatgg
      361 agaaaaaaat cactggatat accaccgttg atatatccca atggcatcgt aaagaacatt
      421 ttgaggcatt tcagtcagtt gctcaatgta cctataacca gaccgttcag ctggatatta
      481 cggccttttt aaagaccgta aagaaaaata agcacaagtt ttatccggcc tttattcaca
      541 ttcttgcccg cctgatgaat gctcatccgg aattccgtat ggcaatgaaa gacggtgagc
      601 tggtgatatg ggatagtgtt cacccttgtt acaccgtttt ccatgagcaa actgaaacgt
      661 tttcatcgct ctggagtgaa taccacgacg atttccggca gtttctacac atatattcgc
      721 aagatgtggc gtgttacggt gaaaacctgg cctatttccc taaagggttt attgagaata
      781 tgtttttcgt ctcagccaat ccctgggtga gtttcaccag ttttgattta aacgtggcca
      841 atatggacaa cttcttcgcc cccgttttca ccatgggcaa atattatacg caaggcgaca
      901 aggtgctgat gccgctggcg attcaggttc atcatgccgt ttgtgatggc ttccatgtcg
      961 gcagaatgct taatgaatta caacagtact gcgatgagtg gcagggcggg gcgtaaacgc
     1021 gtggatccgg cttactaaaa gccagataac agtatgcgta tttgcgcgct gatttttgcg
     1081 gtataagaat atatactgat atgtataccc gaagtatgtc aaaaagaggt atgctatgaa
     1141 gcagcgtatt acagtgacag ttgacagcga cagctatcag ttgctcaagg catatatgat
     1201 gtcaatatct ccggtctggt aagcacaacc atgcagaatg aagcccgtcg tctgcgtgcc
     1261 gaacgctgga aagcggaaaa tcaggaaggg atggctgagg tcgcccggtt tattgaaatg
     1321 aacggctctt ttgctgacga gaacaggggc tggtgaaatg cagtttaagg tttacaccta
     1381 taaaagagag agccgttatc gtctgtttgt ggatgtacag agtgatatta ttgacacgcc
     1441 cgggcgacgg atggtgatcc ccctggccag tgcacgtctg ctgtcagata aagtctcccg
     1501 tgaactttac ccggtggtgc atatcgggga tgaaagctgg cgcatgatga ccaccgatat
     1561 ggccagtgtg ccggtctccg ttatcgggga agaagtggct gatctcagcc accgcgaaaa
     1621 tgacatcaaa aacgccatta acctgatgtt ctggggaata taaatgtcag gctcccttat
     1681 acacagccag tctgcaggtc gaccatagtg actggatatg ttgtgtttta cagtattatg
     1741 tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt
     1801 ctcgttcagc tttcttgtac aaagtggtga tgggcggccg caaaaaaaaa aaaaaaaaaa
     1861 aaaaaaaaaa cctagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgg
     1921 atccgggctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     1981 gcctgaatgg cgaatggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2041 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     2101 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2161 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     2221 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     2281 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     2341 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     2401 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttcctgatg
     2461 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt
     2521 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     2581 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     2641 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     2701 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     2761 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     2821 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     2881 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     2941 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     3001 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     3061 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     3121 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     3181 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     3241 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     3301 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     3361 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     3421 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     3481 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     3541 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     3601 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     3661 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     3721 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     3781 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     3841 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     3901 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     3961 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     4021 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     4081 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     4141 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     4201 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     4261 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     4321 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     4381 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     4441 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     4501 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     4561 gctcacatgg ctcgacagat ct
//
