LOCUS       sequence-429338-        8861 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Helper plasmid for ORBIT in E. coli. Expresses RecT for oligo 
            recombineering and Bxb-1 for pInt incorporation. Includes 
            temperature sensitive origin for plasmid curing..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8861)
  AUTHORS   Saunders SH, Ahmed AM
  TITLE     ORBIT for E. coli: Kilobase-scale oligonucleotide recombineering at 
            high throughput and high efficiency
  JOURNAL   bioRxiv 2023
REFERENCE   2  (bases 1 to 8861)
  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: "bioRxiv 
            2023"
COMMENT     Sequence Label: pHelper_TS_V2_ampR
FEATURES             Location/Qualifiers
     source          1..8861
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1..951)
                     /codon_start=1
                     /gene="rep101 encoding an A56V mutation"
                     /product="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
                     /label=Rep101(Ts)
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      999..1221
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      1700..1727
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(join(1750..2541,2542..2610))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2373..2392
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2611..2715)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2783..2801
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             complement(2967..3845)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     CDS             complement(4066..5031)
                     /codon_start=1
                     /gene="xylS"
                     /product="XylS regulator encoded by the Pseudomonas putida 
                     TOL plasmid pWWO"
                     /label=XylS
                     /note="stimulates transcription from the Pm promoter in the
                     presence of aromatic compounds such as m-toluate or 
                     benzoate"
                     /translation="MDFCLLNEKSQIFVHAEPYAVSDYVNQYVGTHSIRLPKGGRPAGR
                     LHHRIFGCLDLCRISYGGSVRVISPGLETCYHLQIILKGHCLWRGHGQEHYFAPGELLL
                     LNPDDQADLTYSEDCEKFIVKLPSVVLDRACSDNNWHKPREGIRFAARHNLQQLDGFIN
                     LLGLVCDEAEHTKSMPRVQEHYAGIIASKLLEMLGSNVSREIFSKGNPSFERVVQFIEE
                     NLKRNISLERLAELAMMSPRSLYNLFEKHAGTTPKNYIRNRKLESIRACLNDPSANVRS
                     ITEIALDYGFLHLGRFAENYRSAFGELPSDTLRQCKKEVA"
     promoter        5931..6011
                     /label=Pm promoter
                     /note="The bacterial Pm promoter is activated by XylS in 
                     the presence of benzoate or m-toluate (Marques et al., 
                     1999)."
     terminator      6975..7069
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     7072..7091
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             7192..8694
                     /codon_start=1
                     /transl_table=11
                     /gene="Bxb1"
                     /product="Bxb1 integrase"
                     /label=Bxb1 integrase
                     /note="Bxb1 large serine integrase from Mycobacterium 
                     bacteriophage Bxb1 (Ghosh et al. 2003)."
                     /protein_id="AFF61396.1"
                     /translation="MRALVVIRLSRVTDATTSPERQLESCQQLCAQRGWDVVGVAEDLD
                     VSGAVDPFDRKRRPNLARWLAFEEQPFDVIVAYRVDRLTRSIRHLQQLVHWAEDHKKLV
                     VSATEAHFDTTTPFAAVVIALMGTVAQMELEAIKERNRSAAHFNIRAGKYRGSLPPWGY
                     LPTRVDGEWRLVPDPVQRERILEVYHRVVDNHEPLHLVAHDLNRRGVLSPKDYFAQLQG
                     REPQGREWSATALKRSMISEAMLGYATLNGKTVRDDDGAPLVRAEPILTREQLEALRAE
                     LVKTSRAKPAVSTPSLLLRVLFCAVCGEPAYKFAGGGRKHPRYRCRSMGFPKHCGNGTV
                     AMAEWDAFCEEQVLDLLGDAERLEKVWVAGSDSAVELAEVNAELVDLTSLIGSPAYRAG
                     SPQREALDARIAALAARQEELEGLEARPSGWEWRETGQRFGDWWREQDTAAKNTWLRSM
                     NVRLTFDVRGGLTRTIDFGDLQEYEQHLRLGSVVERLHTGMS"
     terminator      8729..8775
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 tcagatcctt ccgtatttag ccagtatgtt ctctagtgtg gttcgttgtt tttgcgtgag
       61 ccatgagaac gaaccattga gatcatactt actttgcatg tcactcaaaa attttgcctc
      121 aaaactggtg agctgaattt ttgcagttaa agcatcgtgt agtgtttttc ttagtccgtt
      181 atgtaggtag gaatctgatg taatggttgt tggtattttg tcaccattca tttttatctg
      241 gttgttctca agttcggtta cgagatccat ttgtctatct agttcaactt ggaaaatcaa
      301 cgtatcagtc gggcggcctc gcttatcaac caccaatttc atattgctgt aagtgtttaa
      361 atctttactt attggtttca aaacccattg gttaagcctt ttaaactcat ggtagttatt
      421 ttcaagcatt aacatgaact taaattcatc aaggctaatc tctatatttg ccttgtgagt
      481 tttcttttgt gttagttctt ttaataacca ctcataaatc ctcatagagt atttgttttc
      541 aaaagactta acatgttcca gattatattt tatgaatttt tttaactgga aaagataagg
      601 caatatctct tcactaaaaa ctaattctaa tttttcgctt gagaacttgg catagtttgt
      661 ccactggaaa atctcaaagc ctttaaccaa aggattcctg atttccacag ttctcgtcat
      721 cagctctctg gttgctttag ctaatacacc ataagcattt tccctactga tgttcatcat
      781 ctgaacgtat tggttataag tgaacgatac cgtccgttct ttccttgtag ggttttcaat
      841 cgtggggttg agtagtgcca cacagcataa aattagcttg gtttcatgct ccgttaagtc
      901 atagcgacta atcgctagtt catttgcttt gaaaacaact aattcagaca tacatctcaa
      961 ttggtctagg tgattttaat cactatacca attgagatgg gctagtcaat gataattact
     1021 agtccttttc ctttgagttg tgggtatctg taaattctgc tagacctttg ctggaaaact
     1081 tgtaaattct gctagaccct ctgtaaattc cgctagacct ttgtgtgttt tttttgttta
     1141 tattcaagtg gttataattt atagaataaa gaaagaataa aaaaagataa aaagaataga
     1201 tcccagccct gtgtataact cactacttta gtcagttccg cagtattaca aaaggatgtc
     1261 gcaaacgctg tttgctcctc tacaaaacag accttaaaac cctaaaggct taagtagcac
     1321 cctcgcaagc tcggttgcgg ccgcaatcgg gcaaatcgct gaatattcct tttgtctccg
     1381 accatcaggc acctgagtcg ctgtcttttt cgtgacattc agttcgctgc gctcacggct
     1441 ctggcagtga atgggggtaa atggcactac aggcgccttt tatggattca tgcaaggaaa
     1501 ctacccataa tacaagaaaa gcccgtcacg ggcttctcag ggcgttttat ggcgggtctg
     1561 ctatgtggtg ctatctgact ttttgctgtt cagcagttcc tgccctctga ttttccagtc
     1621 tgaccacttc ggattatccc gtgacaggtc attcagactg gctaatgcac ccagtaaggc
     1681 agcggtatca tcaacaagca gaaggccatc ctgacggatg gccttttatg agtaaacttg
     1741 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     1801 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     1861 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     1921 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     1981 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     2041 tttgcgcaac gttgttgcca ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat
     2101 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     2161 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     2221 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     2281 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     2341 accgagttgc tcttgcccgg cgtcaacacg ggataatacc gcgccacata gcagaacttt
     2401 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     2461 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     2521 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     2581 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     2641 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     2701 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     2761 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtg tctcatgagc
     2821 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     2881 cgaaaagtgg gtaataactg atataattaa attgaagctc taatttgtga gtttagtata
     2941 catgcattta cttataatac agttttttat gacaacttga cggctacatc attcactttt
     3001 tcttcacaac cggcacggaa ctcgctcggg ctggccccgg tgcatttttt aaatacccgc
     3061 gagaagtaga gttgatcgtc aaaaccaaca ttgcgaccga cggtggcgat aggcatccgg
     3121 gtggtgctca aaagcagctt cgcctggctg atacgttggt cctcgcgcca gcttaagacg
     3181 ctaatcccta actgctggcg gaaaagatgt gacagacgcg acggcgacaa gcaaacatgc
     3241 tgtgcgacgc tggcgatatc aaaattgctg tctgccaggt gatcgctgat gtactgacaa
     3301 gcctcgcgta cccgattatc catcggtgga tggagcgact cgttaatcgc ttccatgcgc
     3361 cgcagtaaca attgctcaag cagatttatc gccagcagct ccgaatagcg cccttcccct
     3421 tgcccggcgt taatgatttg cccaaacagg tcgctgaaat gcggctggtg cgcttcatcc
     3481 gggcgaaaga accccgtatt ggcaaatatt gacggccagt taagccattc atgccagtag
     3541 gcgcgcggac gaaagtaaac ccactggtga taccattcgc gagcctccgg atgacgaccg
     3601 tagtgatgaa tctctcctgg cgggaacagc aaaatatcac ccggtcggca aacaaattct
     3661 cgtccctgat ttttcaccac cccctgaccg cgaatggtga gattgagaat ataacctttc
     3721 attcccagcg gtcggtcgat aaaaaaatcg agataaccgt tggcctcaat cggcgttaaa
     3781 cccgccacca gatgggcatt aaacgagtat cccggcagca ggggatcatt ttgcgcttca
     3841 gccatacttt tcatactccc gccattcaga gaagaaacca attgtccata ttgcatcaga
     3901 cattgccgtc actgcgtctt ttactggctc ttctcgctaa ccaaaccggt aaccccgctt
     3961 attaaaagca ttctgtaaca aagcgggacc aaagccatga caaaaacgcg taacaaaagt
     4021 gtctataatc acggcagaaa agtccacatt gattatttgc acggctcaag ccacttcctt
     4081 tttgcattga cgcagggtgt cggaaggcaa ctcgccgaac gcgctcctat agttttcagc
     4141 gaagcgtccc aaatgtaaga agccgtagtc tagggctatc tcagttatac tacgcacatt
     4201 ggcactggga tcgttcaagc aggcgcggat gctttcgagc ttgcggttgc ggatgtagtt
     4261 cttcggcgtg gtgccggcgt gcttctcgaa caaattgtag agcgagcgtg gactcatcat
     4321 cgccagctcc gctaaccgct caaggctgat attccgtttg agattctcct caatgaattg
     4381 aacgactcgc tcgaaagacg ggttaccttt gctgaaaatt tcacggctga cattgctgcc
     4441 cagcatttcg agcagcttgg aagcgatgat ccccgcatag tgctcttgga cccgaggcat
     4501 cgactttgta tgttccgctt cgtcacaaac taacccgagt agattgataa agccatcgag
     4561 ttgctggaga ttgtgtcgcg cggcgaaacg gataccctcc ctcggcttgt gccaattgtt
     4621 gtcactgcac gcccgatcaa ggaccactga gggcaattta acgataaatt tctcgcaatc
     4681 ttctgaatag gtcaggtcgg cttggtcatc cggattgagc agcaatagtt cgcccggcgc
     4741 aaaatagtgc tcctggccat ggccacgcca caggcaatgg cctttgagta ttatttgcag
     4801 atgataacag gtctctaatc caggcgagat taccctcacg ctaccgccgt agctgattcg
     4861 acacagatcg aggcatccga agattctgtg gtgcagcctg cctgccgggc gcccgccctt
     4921 gggcaggcga atagagtgcg taccgacata ctggttaaca taatcggaga ctgcataggg
     4981 ctcggcgtgg acgaagatct gacttttctc gttcaataag caaaaatcca tagttcacgg
     5041 ttctcttatt ttaatgtggg ctgcttggtg tgatgtagaa aggcgccaag tcgatgaaaa
     5101 tgcatctcga cgtgatgcgt atacgggtta cccccattgc cacgttgcgc catccttttt
     5161 gcaatcagtg accacttttc caagcaaaaa taacgccaag cagaacgaag acgttctttt
     5221 taagaagcga gaacaccaga agttcgtgct gtcggggcat ggggcgacga attggcggat
     5281 aaaggggatc tgctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag
     5341 ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc gtaattacgt
     5401 atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg ttacaccgtt
     5461 ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga cgatttccgg
     5521 cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc
     5581 cctaaagggt ttattgagaa tatgtttttc gtctcagcca atccctgggt gagtttcacc
     5641 agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt caccatgcat
     5701 gggaattagc ttgatctgac caacgaccgg tagcggagct atccaacggc ggtataccag
     5761 gaaaacacac agcaggtaca tcagaacagt accatgactg aagaacaaat agttttttcc
     5821 tgatccataa agcagaacgg cctgctccat gacaaatctg gctccccaac taatgcccca
     5881 tgcagccagc ataaccagca taaagtgcag tgtccggttt gatagggata agtccagcct
     5941 tgcaagaagc ggatacagga gtgcaaaaaa tggctatctc tagtaaggcc taccccttag
     6001 gctttatgca acagaaacaa taataatgga gtcatgacca tgcctaggcc gcgggccgcg
     6061 cgaattcaga aggagaatat accatgaacc aaatcgtgaa gttcactgac gactctggcc
     6121 tggcggttca agttactcca gacgatgttc gccgttatat ctgtgagaac gctactgaaa
     6181 aagaggtggg cctctttctg caactctgtc agactcaacg tctgaatccg tttgtgaaag
     6241 acgcttacct ggtgaaatac ggcggtgctc cagcttctat gattacttcc tatcaagttt
     6301 ttaaccgtcg cgcgtgtcgt gatgctaact atgatggtat caaatctggt gtggttgttc
     6361 tgcgtgacgg tgatgttgtg cataaacgtg gtgctgcgtg ctacaaaaag gcgggtgagg
     6421 agctcatcgg tggttgggcg gaagttcgct ttaaggatgg ccgcgagact gcgtatgctg
     6481 aggtggcgct cgacgactat tccaccggca aatctaattg ggcgaaaatg ccgggtgtta
     6541 tgatcgaaaa atgcgcgaag gctgctgctt ggcgcctcgc gttcccggac acttttcagg
     6601 gcatgtacgc tgcggaggaa atggatcaag cgcaacagcc agaacaggtg cgcgctcagg
     6661 cggagcaacc agtggatctc cagccaatcc gcgaactctt caagccatat tgcgaacact
     6721 tcggcatcac tccggctgag ggtatgactg ctgtttgtgg tgcggtgggc gctgaaggca
     6781 tgcactctat gaccgagcag caagctcgcc gtgctcgcgc ttggatggag gaagaaatgg
     6841 ctgcgccagc tgtggaagcg gagtatgagg ttgttgacga gggcgaggtg ttttaaggat
     6901 cctctagagt cgacctgcag gcatgcaagc ttgcggccgc gtcgtgactg ggaaaaccct
     6961 ggcgactagt cttggactcc tgttgataga tccagtaatg acctcagaac tccatctgga
     7021 tttgttcaga acgctcggtt gccgccgggc gttttttatt ggtgagaatc tatgccatag
     7081 catttttatc cataagatta gcggatccta cctgacgctt tttatcgcaa ctctctactg
     7141 tttctccata cccgtttttt tgggaattcg agctctaagg aggaaaaaca tatgagagcc
     7201 ctggtagtca tccgcctgtc ccgcgtcacc gatgctacga cttcaccgga gcgtcagctg
     7261 gagtcttgcc agcagctctg cgcccagcgc ggctgggacg tcgtcggggt agcggaggat
     7321 ctggacgtct ccggggcggt cgatccgttc gaccggaagc gcagaccgaa cctggcccgg
     7381 tggctagcgt tcgaggagca accgttcgac gtgatcgtgg cgtaccgggt agaccggttg
     7441 acccgatcga tccggcatct gcagcagctg gtccactggg ccgaggacca caagaagctg
     7501 gtcgtctccg cgaccgaagc gcacttcgat acgacgacgc cgtttgcggc ggtcgtcatc
     7561 gcgcttatgg gaacggtggc gcagatggaa ttagaagcga tcaaagagcg gaaccgttcg
     7621 gctgcgcatt tcaatatccg cgccgggaaa taccgaggat ccctgccgcc gtggggatac
     7681 ctgcctacgc gcgtggacgg ggagtggcgg ctggtgccgg accctgtgca gcgagagcgc
     7741 atcctcgagg tgtatcaccg cgtcgtcgac aaccacgagc cgctgcacct ggtggcccac
     7801 gacctgaacc ggcgtggtgt cctgtcgccg aaggactact tcgcgcagct gcaaggccgc
     7861 gagccgcagg gccgggagtg gtcggctacc gcgctgaagc gatcgatgat ctccgaggcg
     7921 atgctcgggt acgcgactct gaacggtaag accgtccgag acgacgacgg agccccgctg
     7981 gtgcgggctg agccgatcct gacccgtgag cagctggagg cgctgcgcgc cgagctcgtg
     8041 aagacctccc gggcgaagcc cgcggtgtct accccgtcgc tgctgctgcg ggtgttgttc
     8101 tgcgcggtgt gcggggagcc cgcgtacaag ttcgccgggg gaggacgtaa gcacccgcgc
     8161 taccgctgcc gctcgatggg gttcccgaag cactgcggga acggcacggt ggcgatggcc
     8221 gagtgggacg cgttctgcga ggagcaggtg ctggatctgc tcggggacgc ggagcgtctg
     8281 gagaaagtct gggtagccgg ctcggactcc gcggtcgaac tcgcggaggt gaacgcggag
     8341 ctggtggacc tgacgtcgct gatcggctcc ccggcctacc gggccggctc tccgcagcga
     8401 gaagcactgg atgcccgtat tgcggcgctg gccgcgcggc aagaggagct ggagggtcta
     8461 gaggctcgcc cgtctggctg ggagtggcgc gagaccgggc agcggttcgg ggactggtgg
     8521 cgggagcagg acaccgcggc aaagaacacc tggcttcggt cgatgaacgt tcggctgacg
     8581 ttcgacgtcc gcggcgggct gactcgcacg atcgacttcg gggatctgca ggagtacgag
     8641 cagcatctca ggctcggcag cgtggtcgaa cggctacaca ccgggatgtc gtaggttaac
     8701 aactagcgta cgatcgactg ccaggcatca aataaaacga aaggctcagt cgaaagactg
     8761 ggcctttcgt tttatgccat catggccgcg ggacgtcggg ggggggggga aagccacgtt
     8821 gtgtctcaaa atctctgatg ttacattgca caagaagaac c
//
