LOCUS       sequence-417957-        5267 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning of PCR products by overlap extension into a gateway entry 
            vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5267)
  AUTHORS   Zare-Mehrjerdi O, Trader G, Kirik V
  TITLE     Overlap extension cloning of PCR products into a Gateway-compatible 
            plasmid vector.
  JOURNAL   Biotechniques. 2023 Jul 9. doi: 10.2144/btn-2023-0001.
  PUBMED    37424091
REFERENCE   2  (bases 1 to 5267)
  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: 
            "Biotechniques. 2023 Jul 9. doi: 10.2144/btn-2023-0001."
COMMENT     Sequence Label: pGATE-1
FEATURES             Location/Qualifiers
     source          1..5267
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(201..220)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(279..298)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(872..1405)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(1594..1599,1600..1616,1617..1622))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     rep_origin      2279..2867
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2768..2787
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3002..3029)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3121..3207)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    3260..3359
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(3704..4009)
                     /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     complement(3806..3825)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(4329..5012)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     4927..4946
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(5013..5115)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(join(5257..5267,1..89))
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 ttgtacaaag tgggcattat aagaaagcat tgcttatcaa tttgttgcaa cgaacaggtc
       61 actatcagtc aaaataaaat cattatttgc catccagctg cagctctggc ccgtgtctca
      121 aaatctctga tgttacattg cacaagataa aaatatatca tcatgaacaa taaaactgtc
      181 tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcgag
      241 gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa
      301 tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg aagcccgatg cgccagagtt
      361 gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact
      421 aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga
      481 tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca gcattccagg tattagaaga
      541 atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca
      601 ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc
      661 gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg
      721 ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc
      781 agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattagg
      841 ccgggaagcc gatctcggct tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa
      901 agtgcatcac ttcttcccgt atgcccaact ttgtatagag agccactgcg ggatcgtcac
      961 cgtaatctgc ttgcacgtag atcacataag caccaagcgc gttggcctca tgcttgagga
     1021 gattgatgag cgcggtggca atgccctgcc tccggtgctc gccggagact gcgagatcat
     1081 agatatagat ctcactacgc ggctgctcaa acttgggcag aacgtaagcc gcgagagcgc
     1141 caacaaccgc ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt
     1201 tcccgaggta atcggagtcc ggctgatgtt gggagtaggt ggctacgtct ccgaactcac
     1261 gaccgaaaag atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg
     1321 tgcgaatgat gcccatactt gagccaccta actttgtttt agggcgactg ccctgctgcg
     1381 taacatcgtt gctgctgcgt aacatcgttg ctgctccata acatcaaaca tcgacccacg
     1441 gcgtaacgcg cttgctgctt ggatgcccga ggcatagact gtacaaaaaa acagtcataa
     1501 caagccatga aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac
     1561 cagttgcgtg agcgcatacg ctacttgcat tacagtttac gaaccgaaca ggcttatgtc
     1621 aactgggttc gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc
     1681 agcgaagtcg aggcatttct gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg
     1741 catcgtcagg cattggcggc cttgctgttc ttctacggca aggtgctgtg cacggatctg
     1801 ccctggcttc aggagatcgg aagacctcgg ccgtcgcggc gcttgccggt ggtgctgacc
     1861 ccggatgaag tggttcgcat cctcggtttt ctggaaggcg agcatcgttt gttcgcccag
     1921 gactctagct atagttctag tggttggcta ctaataggtt gtattgatgt tggacgagtc
     1981 ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct
     2041 ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa
     2101 ttgcagtttc atttgatgct cgatgagttt ttctaatcag aattggttaa ttggttgtaa
     2161 cactggcaga gcattacgct gacttgacgg gacggcgcaa gctcatgacc aaaatccctt
     2221 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     2281 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     2341 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     2401 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     2461 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     2521 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     2581 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     2641 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     2701 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     2761 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     2821 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     2881 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     2941 tatcccctga ttctgtggat aaccgtatta ccgctagcca ggaagagttt gtagaaacgc
     3001 aaaaaggcca tccgtcagga tggccttctg cttagtttga tgcctggcag tttatggcgg
     3061 gcgtcctgcc cgccmccctc cgggccgttg cttcacaacg ttcaaatccg ctcccggcgg
     3121 atttgtccta ctcaggagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc
     3181 ttccgactga gcctttcgtt ttatttgatg cctggcagtt ccctactctc gcgttaacgc
     3241 tagcatggat ctcgggcccc aaataatgat tttattttga ctgatagtga cctgttcgtt
     3301 gcaacaaatt gatgagcaat gcttttttat aatgccaagt ttgtacaaaa aagcaggtcg
     3361 accgacagcc ttccaaatgt tcttctcaaa cggaatcgtc gtatccagcc tactcgctat
     3421 tgtcctcaat gccgtattaa atcataaaaa gaaataagaa aaagaggtgc gagcctcttt
     3481 tttgtgtgac aaaataaaaa catctaccta ttcatatacg ctagtgtcat agtcctgaaa
     3541 atcatctgca tcaagaacaa tttcacaact cttatacttt tctcttacaa gtcgttcggc
     3601 ttcatctgga ttttcagcct ctatacttac taaacgtgat aaagtttctg taatttctac
     3661 tgtatcgacc tgcagactgg ctgtgtataa gggagcctga catttatatt ccccagaaca
     3721 tcaggttaat ggcgtttttg atgtcatttt cgcggtggct gagatcagcc acttcttccc
     3781 cgataacgga gaccggcaca ctggccatat cggtggtcat catgcgccag ctttcatccc
     3841 cgatatgcac caccgggtaa agttcacggg agactttatc tgacagcaga cgtgcactgg
     3901 ccagggggat caccatccgt cgcccgggcg tgtcaataat atcactctgt acatccacaa
     3961 acagacgata acggctctct cttttatagg tgtaaacctt aaactgcatt tcaccagccc
     4021 ctgttctcgt cagcaaaaga gccgttcatt tcaataaacc gggcgacctc agccatccct
     4081 tcctgatttt ccgctttcca gcgttcggca cgcagacgac gggcttcatt ctgcatggtt
     4141 gtgcttacca gaccggagat attgacatca tatatgcctt gagcaactga tagctgtcgc
     4201 tgtcaactgt cactgtaata cgctgcttca tagcatacct ctttttgaca tacttcgggt
     4261 atacatatca gtatatattc ttataccgca aaaatcagcg cgcaaatacg catactgtta
     4321 tctggctttt agtaagccgg atccacgcgg cgtttacgcc ccccctgcca ctcatcgcag
     4381 tactgttgta attcattaag cattctgccg acatggaagc catcacaaac ggcatgatga
     4441 acctgaatcg ccagcggcat cagcaccttg tcgccttgcg tataatattt gcccatggtg
     4501 aaaacggggg cgaagaagtt gtccatattg gccacgttta aatcaaaact ggtgaaactc
     4561 acccagggat tggctgagac gaaaaacata ttctcaataa accctttagg gaaataggcc
     4621 aggttttcac cgtaacacgc cacatcttgc gaatatatgt gtagaaactg ccggaaatcg
     4681 tcgtggtatt cactccagag cgatgaaaac gtttcagttt gctcatggaa aacggtgtaa
     4741 caagggtgaa cactatccca tatcaccagc tcaccgtctt tcattgccat acggaattcc
     4801 ggatgagcat tcatcaggcg ggcaagaatg tgaataaagg ccggataaaa cttgtgctta
     4861 tttttcttta cggtctttaa aaaggccgta atatccagct gaacggtctg gttataggta
     4921 cattgagcaa ctgactgaaa tgcctcaaaa tgttctttac gatgccattg ggatatatca
     4981 acggtggtat atccagtgat ttttttctcc attttagctt ccttagctcc tgaaaatctc
     5041 gataactcaa aaaatacgcc cggtagtgat cttatttcat tatggtgaaa gttggaacct
     5101 cttacgtgcc gatcaacgtc tcattttcgc caaaagttgg cccagggctt cccggtatca
     5161 acagggacac caggatttat ttattctgcg aagtgatctt ccgtcacagg tatttattcg
     5221 gcgcaaagtg cgtcgggtga tgctgccaac ttagtcgacc agctttc
//
