LOCUS       sequence-257561-        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             801..1517
                     /codon_start=1
                     /product="GFP variant optimized for excitation by UV light"
                     /label=GFPuv
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(831..859)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1454..1474
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     promoter        complement(2202..2306)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2374..2392
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(2419..2424,2425..2441,2442..2447)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             2495..3685
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     complement(2847..2866)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             4324..4515
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4509..4531)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4617..4757
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4672..4691
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4772..4789)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4943..5531)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5023..5042)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atcaatctaa agtatatatg agtaaacttg gtctgacagg cttggttgag aatacgccga
       61 agttaaaatc agcccctggc tggcaacggc gtcgatatct ggtgtagggt tacccaccgc
      121 cacgccgcca ccgttaaaac cgacgtccat ccagcccaca tcgtccagca agaaaacaac
      181 cacattcggt ttcttaccgg tttttttctc aagttctgcc agcttctgct gggtttcttt
      241 atcctgcgcc ggatgctgca ttactggcat catattgtcg gcaatagtgg tcgccggttt
      301 aaccagatac tggtttgggt gatcgtatcc ggcaaagcct ttgcgtgcgg tggcagttga
      361 cggggtatct gctgcgctgg ccatgagagg aagagcggcg gcgacagcaa caacaagacg
      421 tttgggtgaa aacgaaaatt ccatgcaaaa tgctccggtt tcatgtcatc aaaatgatga
      481 cgtaattaag cattgataat tgagatccct ctccctgaca ggatgattac ataaataata
      541 gtgacaaaaa taaattattt atttatccag aaaatgaatt ggaaaatcag gagagcgttt
      601 tcaatcctac ctctggcgca gttgatatgt caaacaggtt tatcaaaaag agtattgact
      661 taaagtctaa cctataggat acttacagcc agggagacca caacggtttc ccaagaagga
      721 gatataccat gaccatgatt acgccaagct tgcatgcctg caggtcgact ctagaggatc
      781 cccgggtacc ggtagaaaaa atgagtaaag gagaagaact tttcactgga gttgtcccaa
      841 ttcttgttga attagatggt gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg
      901 aaggtgatgc aacatacgga aaacttaccc ttaaatttat ttgcactact ggaaaactac
      961 ctgttccatg gccaacactt gtcactactt tctcttatgg tgttcaatgc ttttcccgtt
     1021 atccggatca tatgaaacgg catgactttt tcaagagtgc catgcccgaa ggttatgtac
     1081 aggaacgcac tatatctttc aaagatgacg ggaactacaa gacgcgtgct gaagtcaagt
     1141 ttgaaggtga tacccttgtt aatcgtatcg agttaaaagg tattgatttt aaagaagatg
     1201 gaaacattct cggacacaaa ctcgagtaca actataactc acacaatgta tacatcacgg
     1261 cagacaaaca aaagaatgga atcaaagcta acttcaaaat tcgccacaac attgaagatg
     1321 gatccgttca actagcagac cattatcaac aaaatactcc aattggcgat ggccctgtcc
     1381 ttttaccaga caaccattac ctgtcgacac aatctgccct ttcgaaagat cccaacgaaa
     1441 agcgtgacca catggtcctt cttgagtttg taactgctgc tgggattaca catggcatgg
     1501 atgagctcta caaataatga attccaactg agcgccggtc gctaccatta ccaacttgtc
     1561 tggtgtcaaa aataataggc ctactagtcg gccgtacggg cttattatat cgcgttgatt
     1621 attgatgctg tttttagttt taacggcaat taatatatgt gttattaatt gaatgaattt
     1681 tatcattcat aataagtatg tgtaggatca agctcaggtt aaatattcac tcaggaagtt
     1741 attactcagg aagcaaagag gattacagaa ttatctcata acaagtgtta agggatgtta
     1801 tttcccgatt ctctgtggca taataaacga gtagatgctc attccatctc ttatgttcgc
     1861 cttagtgcct cataaactcc ggaatgacgc agagccgttt acggtgctta tcgtccactg
     1921 acagatgtcg cttatgcctc atcagacacc atggacacaa cgttgagtga agcacccact
     1981 tgttgtcata cagacctgtt ttaacgcctg ctccgtaata agagcaggcg tttttttatg
     2041 tatcaggaag gccccggagg tgcttgcctc cgggtgagaa ggaactactg tggcgggtta
     2101 ttctgcaacg ttaacatcaa accgtcgcga cgcatagctg cagcttcttc cggcttgtgc
     2161 agtctgtcca gcgcgtcggc aagccatgcg taatcgtagg cactcttcct ttttcaatat
     2221 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     2281 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     2341 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     2401 cttcaagaat tctcatgttt gacagcttat catcgataag ctttaatgcg gtagtttatc
     2461 acagttaaat tgctaacgca gtcaggcacc gtgtatgaaa tctaacaatg cgctcatcgt
     2521 catcctcggc accgtcaccc tggatgctgt aggcataggc ttggttatgc cggtactgcc
     2581 gggcctcttg cgggatatcg tccattccga cagcatcgcc agtcactatg gcgtgctgct
     2641 agcgctatat gcgttgatgc aatttctatg cgcacccgtt ctcggagcac tgtccgaccg
     2701 ctttggccgc cgcccagtcc tgctcgcttc gctacttgga gccactatcg actacgcgat
     2761 catggcgacc acacccgtcc tgtggatcct ctacgccgga cgcatcgtgg ccggcatcac
     2821 cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg
     2881 ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt
     2941 ggccggggga ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg cggcggtgct
     3001 caacggcctc aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg
     3061 tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat
     3121 gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc
     3181 ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg
     3241 cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg tcactggtcc
     3301 cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg ccgacgcgct
     3361 gggctacgtc ttgctggcgt tcgcgacgcg aggctggatg gccttcccca ttatgattct
     3421 tctcgcttcc ggcggcatcg ggatgcccgc gttgcaggcc atgctgtcca ggcaggtaga
     3481 tgacgaccat cagggacagc ttcaaggatc gctcgcggct cttaccagcc taacttcgat
     3541 cattggaccg ctgatcgtca cggcgattta tgccgcctcg gcgagcacat ggaacgggtt
     3601 ggcatggatt gtaggcgccg ccctatacct tgtctgcctc cccgcgttgc gtcgcggtgc
     3661 atggagccgg gccacctcga cctgaatgga agccggcggc acctcgctaa cggattcacc
     3721 actccaagaa ttggagccaa tcaattcttg cggagaactg tgaatgcgca aaccaaccct
     3781 tggcagaaca tatccatcgc gtccgccatc tccagcagcc gcacgcggcg catctcgggc
     3841 agcgttgggt cctggccacg ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta
     3901 ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga gcgaacgtga
     3961 agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt cttcggtttc
     4021 cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct gcaccattat gttccggatc
     4081 tgcatcgcag gatgctgctg gctaccctgt ggaacaccta catctgtatt aacgaagcgc
     4141 tggcattgac cctgagtgat ttttctctgg tcccgccgca tccataccgc cagttgttta
     4201 ccctcacaac gttccagtaa ccgggcatgt tcatcatcag taacccgtat cgtgagcatc
     4261 ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta cacggaggca
     4321 tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag aagccagaca
     4381 ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga catctgtgaa
     4441 tcgcttcacg accacgctga tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac
     4501 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
     4561 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca
     4621 gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat gcggcatcag
     4681 agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga
     4741 gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4801 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4861 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4921 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4981 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5041 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5101 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5161 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5221 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5281 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5341 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     5401 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5461 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5521 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5581 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5641 taaattaaaa atgaagtttt aa
//
