LOCUS       sequence-129497-        5662 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains 600 bp homology arms to SS9 for one-step markerless genome 
            integration in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5662)
  AUTHORS   Bassalo MC, Garst AD, Halweg-Edwards AL, Grau WC, Domaille DW, 
            Mutalik VK, Arkin AP, Gill RT
  TITLE     Rapid and Efficient One-Step Metabolic Pathway Integration in E. 
            coli.
  JOURNAL   ACS Synth Biol. 2016 Apr 22.
  PUBMED    27072506
REFERENCE   2  (bases 1 to 5662)
  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: "ACS Synth 
            Biol. 2016 Apr 22."
COMMENT     Sequence Label: pSS9
FEATURES             Location/Qualifiers
     source          1..5662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1296..2012
                     /codon_start=1
                     /product="GFP variant optimized for excitation by UV light"
                     /label=GFPuv
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(1326..1354)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1949..1969
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     promoter        complement(2697..2801)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2869..2887
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(2914..2919,2920..2936,2937..2942)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             2990..4180
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSITGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     complement(3342..3361)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             4819..5010
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5004..5026)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5112..5252
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5167..5186
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5267..5284)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(5438..5662,1..364))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5518..5537)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
       61 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
      121 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
      181 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
      241 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
      301 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
      361 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
      421 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
      481 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acaggcttgg
      541 ttgagaatac gccgaagtta aaatcagccc ctggctggca acggcgtcga tatctggtgt
      601 agggttaccc accgccacgc cgccaccgtt aaaaccgacg tccatccagc ccacatcgtc
      661 cagcaagaaa acaaccacat tcggtttctt accggttttt ttctcaagtt ctgccagctt
      721 ctgctgggtt tctttatcct gcgccggatg ctgcattact ggcatcatat tgtcggcaat
      781 agtggtcgcc ggtttaacca gatactggtt tgggtgatcg tatccggcaa agcctttgcg
      841 tgcggtggca gttgacgggg tatctgctgc gctggccatg agaggaagag cggcggcgac
      901 agcaacaaca agacgtttgg gtgaaaacga aaattccatg caaaatgctc cggtttcatg
      961 tcatcaaaat gatgacgtaa ttaagcattg ataattgaga tccctctccc tgacaggatg
     1021 attacataaa taatagtgac aaaaataaat tatttattta tccagaaaat gaattggaaa
     1081 atcaggagag cgttttcaat cctacctctg gcgcagttga tatgtcaaac aggtttatca
     1141 aaaagagtat tgacttaaag tctaacctat aggatactta cagccaggga gaccacaacg
     1201 gtttcccaag aaggagatat accatgacca tgattacgcc aagcttgcat gcctgcaggt
     1261 cgactctaga ggatccccgg gtaccggtag aaaaaatgag taaaggagaa gaacttttca
     1321 ctggagttgt cccaattctt gttgaattag atggtgatgt taatgggcac aaattttctg
     1381 tcagtggaga gggtgaaggt gatgcaacat acggaaaact tacccttaaa tttatttgca
     1441 ctactggaaa actacctgtt ccatggccaa cacttgtcac tactttctct tatggtgttc
     1501 aatgcttttc ccgttatccg gatcatatga aacggcatga ctttttcaag agtgccatgc
     1561 ccgaaggtta tgtacaggaa cgcactatat ctttcaaaga tgacgggaac tacaagacgc
     1621 gtgctgaagt caagtttgaa ggtgataccc ttgttaatcg tatcgagtta aaaggtattg
     1681 attttaaaga agatggaaac attctcggac acaaactcga gtacaactat aactcacaca
     1741 atgtatacat cacggcagac aaacaaaaga atggaatcaa agctaacttc aaaattcgcc
     1801 acaacattga agatggatcc gttcaactag cagaccatta tcaacaaaat actccaattg
     1861 gcgatggccc tgtcctttta ccagacaacc attacctgtc gacacaatct gccctttcga
     1921 aagatcccaa cgaaaagcgt gaccacatgg tccttcttga gtttgtaact gctgctggga
     1981 ttacacatgg catggatgag ctctacaaat aatgaattcc aactgagcgc cggtcgctac
     2041 cattaccaac ttgtctggtg tcaaaaataa taggcctact agtcggccgt acgggcttat
     2101 tatatcgcgt tgattattga tgctgttttt agttttaacg gcaattaata tatgtgttat
     2161 taattgaatg aattttatca ttcataataa gtatgtgtag gatcaagctc aggttaaata
     2221 ttcactcagg aagttattac tcaggaagca aagaggatta cagaattatc tcataacaag
     2281 tgttaaggga tgttatttcc cgattctctg tggcataata aacgagtaga tgctcattcc
     2341 atctcttatg ttcgccttag tgcctcataa actccggaat gacgcagagc cgtttacggt
     2401 gcttatcgtc cactgacaga tgtcgcttat gcctcatcag acaccatgga cacaacgttg
     2461 agtgaagcac ccacttgttg tcatacagac ctgttttaac gcctgctccg taataagagc
     2521 aggcgttttt ttatgtatca ggaaggcccc ggaggtgctt gcctccgggt gagaaggaac
     2581 tactgtggcg ggttattctg caacgttaac atcaaaccgt cgcgacgcat agctgcagct
     2641 tcttccggct tgtgcagtct gtccagcgcg tcggcaagcc atgcgtaatc gtaggcactc
     2701 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     2761 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     2821 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     2881 acgaggccct ttcgtcttca agaattctca tgtttgacag cttatcatcg ataagcttta
     2941 atgcggtagt ttatcacagt taaattgcta acgcagtcag gcaccgtgta tgaaatctaa
     3001 caatgcgctc atcgtcatcc tcggcaccgt caccctggat gctgtaggca taggcttggt
     3061 tatgccggta ctgccgggcc tcttgcggga tatcgtccat tccgacagca tcgccagtca
     3121 ctatggcgtg ctgctagcgc tatatgcgtt gatgcaattt ctatgcgcac ccgttctcgg
     3181 agcactgtcc gaccgctttg gccgccgccc agtcctgctc gcttcgctac ttggagccac
     3241 tatcgactac gcgatcatgg cgaccacacc cgtcctgtgg atcctctacg ccggacgcat
     3301 cgtggccggc atcaccggcg ccacaggtgc ggttgctggc gcctatatcg ccgacatcac
     3361 cgatggggaa gatcgggctc gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat
     3421 ggtggcaggc cccgtggccg ggggactgtt gggcgccatc tccttgcatg caccattcct
     3481 tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa tgcaggagtc
     3541 gcataaggga gagcgtcgac cgatgccctt gagagccttc aacccagtca gctccttccg
     3601 gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact
     3661 cgtaggacag gtgccggcag cgctctgggt cattttcggc gaggaccgct ttcgctggag
     3721 cgcgacgatg atcggcctgt cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc
     3781 cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag caggccatta tcgccggcat
     3841 ggcggccgac gcgctgggct acgtcttgct ggcgttcgcg acgcgaggct ggatggcctt
     3901 ccccattatg attcttctcg cttccggcgg catcgggatg cccgcgttgc aggccatgct
     3961 gtccaggcag gtagatgacg accatcaggg acagcttcaa ggatcgctcg cggctcttac
     4021 cagcctaact tcgatcactg gaccgctgat cgtcacggcg atttatgccg cctcggcgag
     4081 cacatggaac gggttggcat ggattgtagg cgccgcccta taccttgtct gcctccccgc
     4141 gttgcgtcgc ggtgcatgga gccgggccac ctcgacctga atggaagccg gcggcacctc
     4201 gctaacggat tcaccactcc aagaattgga gccaatcaat tcttgcggag aactgtgaat
     4261 gcgcaaacca acccttggca gaacatatcc atcgcgtccg ccatctccag cagccgcacg
     4321 cggcgcatct cgggcagcgt tgggtcctgg ccacgggtgc gcatgatcgt gctcctgtcg
     4381 ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga atcaccgata
     4441 cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc aacaacatga
     4501 atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc
     4561 attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac acctacatct
     4621 gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg ccgcatccat
     4681 accgccagtt gtttaccctc acaacgttcc agtaaccggg catgttcatc atcagtaacc
     4741 cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa cagaaatccc
     4801 ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt
     4861 atcagaagcc agacattaac gcttctggag aaactcaacg agctggacgc ggatgaacag
     4921 gcagacatct gtgaatcgct tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg
     4981 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     5041 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     5101 gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta
     5161 actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc
     5221 acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact
     5281 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     5341 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     5401 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     5461 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     5521 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     5581 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     5641 gctgtaggta tctcagttcg gt
//
